Production of sialylated n-glycans in lower eukaryotes

ABSTRACT

The present invention relates to eukaryotic host cells which have been modified to produce sialylated glycoproteins by the heterologous expression of a set of glycosyltransferases, including sialyltransferase and/or trans-sialidase, to become host-strains for the production of mammalian, e.g., human therapeutic glycoproteins. Novel eukaryotic host cells expressing a CMP-sialic acid biosynthetic pathway for the production of sialylated glycoproteins are also provided. The invention provides nucleic acid molecules and combinatorial libraries which can be used to successfully target and express mammalian enzymatic activities (such as those involved in sialylation) to intracellular compartments in a eukaryotic host cell. The process provides an engineered host cell which can be used to express and target any desirable gene(s) involved in glycosylation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part (“CIP”) of three applications: (1) U.S. application Ser. No. 10/371,877, filed Feb. 20, 2003, which is a CIP of U.S. application Ser. No. 09/892,591, filed Jun. 27, 2001, now issued as U.S. Pat. No. 7,029,872 which claims the benefit of U.S. Provisional Application Ser. Nos. 60/214,358, filed Jun. 28, 2000; 60/215,638, filed Jun. 30, 2000; and 60/279,997, filed Mar. 30, 2001; (2) U.S. application Ser. No. 11/084,624, filed Mar. 17, 2005, which claims the benefit of U.S. Provisional Application No. 60/554,139, filed Mar. 17, 2004; and (3) U.S. application Ser. No. 11/108,088, filed Apr. 15, 2005, which is: (a) a CIP of U.S. application Ser. No. 10/371,877, filed Feb. 20, 2003, which is a CIP of U.S. application Ser. No. 09/892,591, filed Jun. 27, 2001, which claims the benefit of U.S. Provisional Application Nos. 60/214,358, filed Jun. 28, 2000; 60/215,638, filed Jun. 30, 2000; and 60/279,997, filed Mar. 30, 2001; (b) a CIP of PCT/US02/41510, filed Dec. 24, 2002, which claims the benefit of U.S. Provisional Application No. 60/344,169, filed Dec. 27, 2001; and (c) claims the benefit of U.S. Provisional Application No. 60/562,424, filed Apr. 15, 2004. Each of the above cited references is incorporated herein by applications in its entirety.

FIELD OF THE INVENTION

The present invention is directed to methods and compositions by which non-human eukaryotic host cells, such as fungi or other eukaryotic cells, can be genetically modified to produce glycosylated proteins (glycoproteins) having patterns of glycosylation similar to those of glycoproteins produced by animal cells, especially human cells, which are useful as human or animal therapeutic agents. In particular this application relates to methods and compositions for the production of sialylated glycoproteins in non-human eukaryotic host cells that do not normally produce sialylated glycoproteins.

BACKGROUND OF THE INVENTION Glycosylation Pathways in Humans and Lower Eukaryotes

After DNA is transcribed and translated into a protein, further post-translational processing involves the attachment of sugar residues, a process known as glycosylation. Different organisms produce different glycosylation enzymes (glycosyltransferases and glycosidases), and have different substrates (nucleotide sugars) available, so that the glycosylation patterns as well as composition of the individual oligosaccharides, even of the same protein, will be different depending on the host system in which the particular protein is being expressed. Bacteria typically do not glycosylate proteins, and if so only in a very unspecific manner (Moens, 1997). (Throughout the specification, scientific publications will be cited by reference to the senior author and publication year; complete citations are found at the end of the specification.) Lower eukaryotes such as filamentous fungi and yeast add primarily mannose and mannosylphosphate sugars. The resulting glycan is known as a “high-mannose” type glycan or a mannan. Plant cells and insect cells (such as Sf9 cells) glycosylate proteins in yet another way. By contrast, in higher eukaryotes such as humans, the nascent oligosaccharide side chain may be trimmed to remove several mannose residues and elongated with additional sugar residues that typically are not found in the N-glycans of lower eukaryotes. See, e.g., Bretthauer, 1999; Martinet, 1998; Weikert, 1999; Malissard, 2000; Jarvis 1998; and Takeuchi, 1997.

Synthesis of a mammalian-type oligosaccharide structure begins with a set of sequential reactions in the course of which sugar residues are added and removed while the protein moves along the secretory pathway in the host organism. The enzymes which reside along the glycosylation pathway of the host organism or cell determine the resulting glycosylation patterns of secreted proteins. Thus, the resulting glycosylation pattern of proteins expressed in lower eukaryotic host cells differs substantially from the glycosylation pattern of proteins expressed in higher eukaryotes such as humans and other mammals (Bretthauer, 1999). The structure of a typical fungal N-glycan is shown in FIG. 1A.

The early steps of human glycosylation can be divided into at least two different phases: (i) lipid-linked Glc₃Man₉GlcNAc₂ oligosaccharides are assembled by a sequential set of reactions at the membrane of the endoplasmic reticulum (ER) and (ii) the transfer of this oligosaccharide from the lipid anchor dolichyl pyrophosphate onto de novo synthesized protein. The site of the specific transfer is defined by an asparagine (Asn) residue in the sequence Asn-Xaa-Ser/Thr where Xaa can be any amino acid except proline (Gavel, 1990). Further processing by glucosidases and mannosidases occurs in the ER before the nascent glycoprotein is transferred to the early Golgi apparatus, where additional mannose residues are removed by Golgi specific alpha (α)-1,2-mannosidases. Processing continues as the protein proceeds through the Golgi. In the medial Golgi, a number of modifying enzymes, including N-acetylglucosaminyl Transferases (GnTI, GnTII, GnTIII, GnTIV and GnTV), mannosidase II and fucosyltransferases, add and remove specific sugar residues. Finally, in the trans-Golgi, galactosyltransferases (GalT) and sialyltransferases (ST) produce a glycoprotein structure that is released from the Golgi. It is this structure, characterized by bi-, tri- and tetra-antennary structures, containing galactose, fucose, N-acetylglucosamine and a high degree of terminal sialic acid, that gives glycoproteins their human characteristics. The structure of a typical human N-glycan is shown in FIG. 1B.

In nearly all eukaryotes, glycoproteins are derived from a common lipid-linked oligosaccharide precursor Glc₃Man₉GlcNAc₂-dolichol-pyrophosphate. Within the endoplasmic reticulum, synthesis and processing of dolichol pyrophosphate bound oligosaccharides are identical between all known eukaryotes. However, further processing of the core oligosaccharide by fungal cells, e.g., yeast, once it has been transferred to a peptide leaving the ER and entering the Golgi, differs significantly from humans as it moves along the secretory pathway and involves the addition of several mannose sugars.

In yeast, these steps are catalyzed by Golgi residing mannosyltransferases, like Och1p, Mnt1p and Mnn1p, which sequentially add mannose sugars to the core oligosaccharide. The resulting structure is undesirable for the production of human-like proteins and it is thus desirable to reduce or eliminate mannosyltransferase activity. Mutants of Saccharomyces cerevisiae (S. cerevisiae), deficient in mannosyltransferase activity (for example och1 or mnn9 mutants) have been shown to be non-lethal and display reduced mannose content in the oligosaccharide of yeast glycoproteins. Other oligosaccharide processing enzymes, such as mannosylphosphate transferase, may also have to be eliminated depending on the host's particular endogenous glycosylation pattern.

Sugar Nucleotide Precursors

The N-glycans of animal glycoproteins typically include galactose, fucose, and terminal sialic acid. These sugars are not found on glycoproteins produced in yeast and filamentous fungi. In humans and other non-human eukaryotic cells, the full range of sugar nucleotide precursors (e.g. UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine, CMP-N-acetylneuraminic acid, UDP-galactose, GDP-fucose, etc.) are synthesized in the cytosol and transported into the Golgi, where they are attached to the core oligosaccharide by glycosyltransferases. (Sommers, 1981; Sommers, 1982; Perez, 1987).

Glycosyl transfer reactions typically yield a side product which is a nucleoside diphosphate or monophosphate. While monophosphates can be directly exported in exchange for nucleoside triphosphate sugars by an antiport mechanism, diphosphonucleosides (e.g. GDP) have to be cleaved by phosphatases (e.g. GDPase) to yield nucleoside monophosphates and inorganic phosphate prior to being exported. This reaction is important for efficient glycosylation; for example, GDPase from Saccharomyces cerevisiae (S. cerevisiae) has been found to be necessary for mannosylation. However that GDPase has 90% reduced activity toward UDP (Berninsone, 1994). Lower eukaryotes typically lack UDP-specific diphosphatase activity in the Golgi since they do not utilize UDP-sugar precursors for Golgi-based glycoprotein synthesis. Schizosaccharomyces pombe, a yeast found to add galactose residues to cell wall polysaccharides (from UDP-galactose) has been found to have specific UDPase activity, indicating the potential requirement for such an enzyme (Berninsone, 1994). UDP is known to be a potent inhibitor of glycosyltransferases and the removal of this glycosylation side product may be important to prevent glycosyl-transferase inhibition in the lumen of the Golgi (Khatara, 1974). See Berninsone, 1995; Beaudet, 1998.

Sequential Processing of N-Glycans by Compartmentalized Enzyme Activities

Sugar transferases and glycosidases (e.g., mannosidases) line the inner (luminal) surface of the ER and Golgi apparatus and thereby provide a “catalytic” surface that allows for the sequential processing of glycoproteins as they proceed through the ER and Golgi network. The multiple compartments of the cis, medial, and trans Golgi and the trans-Golgi Network (TGN), provide the different localities in which the ordered sequence of glycosylation reactions can take place. As a glycoprotein proceeds from synthesis in the ER to full maturation in the late Golgi or TGN, it is sequentially exposed to different glycosidases, mannosidases and glycosyltransferases such that a specific carbohydrate structure may be synthesized. Much work has been dedicated to revealing the exact mechanism by which these enzymes are retained and anchored to their respective organelle. The evolving picture is complex but evidence suggests that stem region, membrane spanning region and cytoplasmic tail, individually or in concert, direct enzymes to the membrane of individual organelles and thereby localize the associated catalytic domain to that locus (see, e.g., Gleeson, 1998).

In some cases, these specific interactions were found to function across species. For example, the membrane spanning domain of α2,6-ST from rats, an enzyme known to localize in the trans-Golgi of the animal, was shown to also localize a reporter gene (invertase) in the yeast Golgi (Schwientek, 1995).

However, the very same membrane spanning domain as part of a full-length α2,6-ST was retained in the ER and not further transported to the Golgi of yeast (Krezdorn, 1994). A full length GalT from humans was not even synthesized in yeast, despite demonstrably high transcription levels. In contrast, the transmembrane region of the same human GalT fused to an invertase reporter was able to direct localization to the yeast Golgi, albeit it at low production levels. Schwientek and co-workers have shown that fusing 28 amino acids of a yeast mannosyltransferase (MNT1), a region containing a cytoplasmic tail, a transmembrane region and eight amino acids of the stem region, to the catalytic domain of human GalT are sufficient for Golgi localization of an active GalT. Other galactosyltransferases appear to rely on interactions with enzymes resident in particular organelles because, after removal of their transmembrane region, they are still able to localize properly.

Improper localization of a glycosylation enzyme may prevent proper functioning of the enzyme in the pathway. For example, Aspergillus nidulans, which has numerous α-1,2-mannosidases (Eades, 2000), does not add GlcNAc to Man₅GlcNAc₂ when transformed with the rabbit GnTI gene, despite a high overall level of GnTI activity (Kalsner et al., 1995). GnTI, although actively expressed, may be incorrectly localized such that the enzyme is not in contact with both of its substrates: UDP-GlcNAc and a productive Man₅GlcNAc₂ substrate (not all Man₅GlcNAc₂ structures are productive; see below). Alternatively, the host organism may not provide an adequate level of UDP-GlcNAc in the Golgi or the enzyme may be properly localized but nevertheless inactive in its new environment. In addition, Man₅GlcNAc₂ structures present in the host cell may differ in structure from Man₅GlcNAc₂ found in mammals. Maras and coworkers found that about one third of the N-glycans from cellobiohydrolase I (CBHI) obtained from T. reesei can be trimmed to Man₅GlcNAc₂ by A. saitoi 1,2 mannosidase in vitro. Fewer than 1% of those N-glycans, however, could serve as a productive substrate for GnTI. The mere presence of Man₅GlcNAc₂, therefore, does not assure that further processing to Man₅GlcNAc₂ can be achieved. It is formation of a productive, GnTI-reactive Man₅GlcNAc₂ structure that is required. Although Man₅GlcNAc₂ could be produced in the cell (about 27 mol %), only a small fraction could be converted to Man₅GlcNAc₂ (less than about 5%, see WO 01/14522).

To date, there is no reliable way of predicting whether a particular heterologously expressed glycosyltransferase or mannosidase in a lower eukaryote will be (1), sufficiently translated (2), catalytically active or (3) located to the proper organelle within the secretory pathway. Because all three of these are necessary to affect glycosylation patterns in lower eukaryotes, a systematic scheme to achieve the desired catalytic function and proper retention of enzymes in the absence of predictive tools, which are currently not available, would be desirable.

Production of Therapeutic Glycoproteins

A significant number of proteins isolated from humans or animals are post-translationally modified, with glycosylation being one of the most significant modifications. An estimated 70% of all therapeutic proteins are glycosylated and thus currently rely on a production system (i.e., host cell) that is able to glycosylate in a manner similar to humans. Several studies have shown that glycosylation plays an important role in determining the (1) immunogenicity, (2) pharmacokinetic properties, (3) trafficking, and (4) efficacy of therapeutic proteins. It is thus not surprising that substantial efforts by the pharmaceutical industry have been directed at developing processes to obtain glycoproteins that are as “humanoid” or “human-like” as possible. To date, most glycoproteins are made in a mammalian host system. This may involve the genetic engineering of such mammalian cells to enhance the degree of sialylation (i.e., terminal addition of sialic acid) of proteins expressed by the cells, which is known to improve pharmacokinetic properties of such proteins. Alternatively, one may improve the degree of sialylation by in vitro addition of such sugars using known glycosyltransferases and their respective nucleotide sugars (e.g., 2,3-sialyltransferase and CMP-sialic acid).

While most higher eukaryotes carry out glycosylation reactions that are similar to those found in humans, recombinant human proteins expressed in the above mentioned host systems invariably differ from their “natural” human counterpart (Raju, 2000). Extensive development work has thus been directed at finding ways to improve the “human character” of proteins made in these expression systems. This includes the optimization of fermentation conditions and the genetic modification of protein expression hosts by introducing genes encoding enzymes involved in the formation of human-like glycoforms (Werner, 1998; Weikert, 1999; Andersen, 1994; Yang, 2000). Inherent problems associated with all mammalian expression systems have not been solved.

Most, if not all, currently produced therapeutic glycoproteins are therefore expressed in mammalian cells and much effort has been directed at improving (i.e., “humanizing”) the glycosylation pattern of these recombinant proteins. Changes in medium composition as well as the co-expression of genes encoding enzymes involved in human glycosylation have been successfully employed (see, for example, Weikert, 1999).

Glycoprotein Production Using Eukaryotic Microorganisms

The lack of a suitable mammalian expression system is a significant obstacle to the low-cost and safe production of recombinant human glycoproteins for therapeutic applications. It would be desirable to produce recombinant proteins similar to their mammalian, e.g., human, counterparts in lower eukaryotes (fungi and yeast). Production of glycoproteins via the fermentation of microorganisms would offer numerous advantages over existing systems. Although the core oligosaccharide structure transferred to a protein in the endoplasmic reticulum is basically identical in mammals and lower eukaryotes, substantial differences have been found in the subsequent processing reactions which occur in the Golgi apparatus of fungi and mammals. In fact, even amongst different lower eukaryotes there exist a great variety of glycosylation structures. This has historically prevented the use of lower eukaryotes as hosts for the production of recombinant human glycoproteins despite otherwise notable advantages over mammalian expression systems.

Therapeutic glycoproteins produced in a microorganism host such as yeast utilizing the endogenous host glycosylation pathway differ structurally from those produced in mammalian cells and typically show greatly reduced therapeutic efficacy. Such glycoproteins are typically immunogenic in humans and show a reduced half-life (and thus bioactivity) in vivo after administration (Takeuchi, 1997). Specific receptors in humans and animals (i.e., macrophage mannose receptors) can recognize terminal mannose residues and promote the rapid clearance of the foreign glycoprotein from the bloodstream. Additional adverse effects may include changes in protein folding, solubility, susceptibility to proteases, trafficking, transport, compartmentalization, secretion, recognition by other proteins or factors, antigenicity, or allergenicity.

Yeast and filamentous fungi have both been successfully used for the production of recombinant proteins, both intracellular and secreted (Cereghino, 2000; Harkki, 1989; Berka, 1992; Svetina, 2000). Various yeasts, such as K. lactis, Pichia pastoris, Pichia methanolica, and Hansenula polymorpha, have played particularly important roles as eukaryotic expression systems because they are able to grow to high cell densities and secrete large quantities of recombinant protein. Likewise, filamentous fungi, such as Aspergillus niger, Fusarium sp., Neurospora crassa and others, have been used to efficiently produce glycoproteins at the industrial scale. However, as noted above, glycoproteins expressed in any of these eukaryotic microorganisms differ substantially in N-glycan structure from those in animals. This has prevented the use of yeast or filamentous fungi as hosts for the production of many therapeutic glycoproteins.

Although glycosylation in yeast and fungi is very different than in humans, some common elements are shared. The first step, the transfer of the core oligosaccharide structure to the nascent protein, is highly conserved in all eukaryotes including yeast, fungi, plants and humans (compare FIGS. 1A and 1B). Subsequent processing of the core oligosaccharide, however, differs significantly in yeast and involves the addition of several mannose sugars. This step is catalyzed by mannosyltransferases residing in the Golgi (e.g. OCH1, MNT1, MNN1, etc.), which sequentially add mannose sugars to the core oligosaccharide. The resulting structure is undesirable for the production of humanoid proteins and it is thus desirable to reduce or eliminate mannosyltransferase activity. Mutants of S. cerevisiae deficient in mannosyltransferase activity (e.g. och1 or mnn9 mutants) have shown to be non-lethal and display a reduced mannose content in the oligosaccharide of yeast glycoproteins. Other oligosaccharide processing enzymes, such as mannosylphosphate transferase, may also have to be eliminated depending on the host's particular endogenous glycosylation pattern. After reducing undesired endogenous glycosylation reactions, the formation of complex N-glycans has to be engineered into the host system. This requires the stable expression of several enzymes and sugar-nucleotide transporters. Moreover, one has to localize these enzymes so that a sequential processing of the maturing glycosylation structure is ensured.

Several efforts have been made to modify the glycosylation pathways of eukaryotic microorganisms to provide glycoproteins more suitable for use as mammalian therapeutic agents. However, N-glycans resembling those made in human cells (e.g., with complex or hybrid glycan structures) were not obtained.

Yeasts produce a variety of mannosyltransferases (e.g., 1,3-mannosyltransferases such as MNN1 in S. cerevisiae; Graham, 1991, 1,2-mannosyltransferases (e.g. KTR/KRE family from S. cerevisiae), 1,6-mannosyltransferases (e.g., OCH1 from S. cerevisiae), mannosylphosphate transferases and their regulators (e.g., MNN4 and MNN6 from S. cerevisiae) and additional enzymes that are involved in endogenous glycosylation reactions. Many of these genes have been deleted individually giving rise to viable organisms having altered glycosylation profiles.

While it is useful to engineer strains that are able to produce Man₅GlcNAc₂ as the primary N-glycan structure, any attempt to further modify these high mannose precursor structures to more closely resemble human glycans requires additional in vivo or in vitro steps. Methods to further humanize glycans from fungal and yeast sources in vitro are described in U.S. Pat. No. 5,834,251. As discussed above, however, if Man₅GlcNAc₂ is to be further humanized in vivo, one has to ensure that the generated Man₅GlcNAc₂ structures are, in fact, generated intracellularly and not the product of mannosidase activity in the medium. Complex N-glycan formation in yeast or fungi will require high levels of Man₅GlcNAc₂ to be generated within the cell because only intracellular Man₅GlcNAc₂ glycans can be further processed to hybrid and complex N-glycans in vivo. In addition, one has to demonstrate that the majority of Man₅GlcNAc₂ structures generated are in fact a substrate for GnTI and thus allow the formation of hybrid and complex N-glycans.

Accordingly, the need exists for methods to produce glycoproteins characterized by a high intracellular Man₅GlcNAc₂ content which can be further processed into human-like glycoprotein structures in non-human eukaryotic host cells, and particularly in yeast and filamentous fungi.

Addition of Sialic Acid to N-Glycans

Sialic acids (Sia) are a unique group of N- or O-substituted derivatives of N-acetylneuraminic acid (Neu5Ac) which are ubiquitous in animals of the deuterostome lineage, from starfish to humans. In other organisms, including most plants, protists, Archaea, and eubacteria, these compounds are thought to be absent (Warren, 1994). Exceptions have been identified, all of which are in pathogenic organisms, including certain bacteria, protozoa and fungi (Kelm, 1997; Parodi, 1993; Alviano, 1999). The mechanism by which pathogenic fungi, including Cryptococcus neoformans and Candida albicans, acquire sialic acid on cell surface glycoproteins and glycolipids remains undetermined (Alviano, 1999). When these organisms are grown in sialic acid-free media, sialic acid residues are found on cellular glycans, suggesting de novo synthesis of sialic acid. To date, no enzymes have been identified in fungi that are involved in the biosynthesis of sialic acid. The mechanism by which protozoa sialylate cell surface glycans has been well-characterized. Protozoa, such as Trypanosoma cruzi, possess an external trans-sialidase that adds sialic acid to cell surface glycoproteins and glycolipids in a CMP-Sia independent mechanism (Parodi, 1993). The identification of a similar trans-sialidase in fungi would help to elucidate the mechanism of sialic acid transfer on cellular glycans, but such a protein has not yet been identified or isolated.

Despite the absence and/or ambiguity of sialic acid biosynthesis in fungi, sialic acid biosynthesis in pathogenic bacteria and mammalian cells is well understood. A group of pathogenic bacteria have been identified that synthesize sialic acids de novo to generate sialylated glycolipids that occur on the cell surface (Vimr, 1995). Although sialic acids on the surface of these pathogenic organisms are predominantly thought to be a means of evading the host immune system, these same sialic acid molecules are also involved in many processes in higher organisms, including protein targeting, cell-cell interaction, cell-substrate recognition and adhesion (Schauer, 2000).

The presence of sialic acids can affect biological activity and half-life of glycoproteins in vivo (MacDougall, 1999). For example, the importance of sialic acids has been demonstrated in studies of human erythropoietin (hEPO). The terminal sialic acid residues on the carbohydrate chains of the N-linked glycan of this glycoprotein prevent rapid clearance of hEPO from the blood and improve in vivo activity. Asialylated-hEPO (asialo-hEPO), which terminates in a galactose residue, has dramatically decreased erythropoietic activity in vivo. This decrease is caused by the increased clearance of the asialo-hEPO by the hepatic asialoglycoprotein receptor (Fukuda, 1989; Spivak, 1989; Spivak, 1989). Similarly, the absence of terminal sialic acid on many therapeutic glycoproteins can reduce efficacy in vivo, and thus require more frequent patient dosing regimes.

SUMMARY OF THE INVENTION

Host cells and cell lines having genetically modified glycosylation pathways that allow them to carry out a specified sequence of enzymatic reactions which mimic the processing of glycoproteins in mammals, especially in humans, have been developed. Recombinant proteins expressed in these engineered hosts yield glycoproteins more similar, if not substantially identical, to their mammalian, e.g., human counterparts. Moreover, substantially homogeneous glycoprotein populations having particular desired glycan structures may be produced. Host cells of the invention, e.g., lower eukaryotic micro-organisms and other non-human, eukaryotic host cells grown in culture, are modified to produce N-glycans produced along human glycosylation pathways. This is achieved using a combination of engineering and/or selection of strains that: (1) do not express certain enzymes which create the undesirable structures characteristic of the fungal glycoproteins; (2) express heterologous enzymes selected either to have optimal activity under the conditions present in the host cell where activity is to be achieved; or (3) combinations thereof; wherein the genetically engineered host cell expresses at least one heterologous enzyme activity required to produce a “human-like” glycoprotein. Host cells of the invention may be modified further by heterologous expression of one or more activities such as glycosyltransferases, glycosidase (such as mannosidases), sugar nucleotide transporters, and the like, to become strains for the production of mammalian, e.g., human therapeutic glycoproteins.

The present invention thus provides a glycoprotein production method using (1) a lower eukaryotic host such as a unicellular or filamentous fungus, or (2) any eukaryotic organism, e.g., a non-human eukaryotic cell or organism that has a different glycosylation pattern from humans, to modify the glycosylation composition and structures of the proteins made in a host organism (“host cell”) so that they resemble more closely carbohydrate structures found in mammalian, e.g., human proteins. The process allows one to obtain an engineered host cell in which desirable gene(s), e.g., one(s) involved in glycosylation, is(are) expressed and its (their) product(s) targeted to a subcellular location in the host cell by methods that are well-established in the scientific literature and generally known to the artisans in the field of protein expression. For the production of therapeutic proteins, this method may be adapted to engineer cell lines in which any desired glycosylation structure may be obtained on proteins expressed in the engineered cells.

In one embodiment, N-glycans made in the host cells have a Man₅GlcNAc₂ core structure which may then be modified further by heterologous expression of one or more enzymes, e.g., glycosyltransferases, glycosidases such as mannosidases, sugar transporters and the like, to yield modified glycoproteins, e.g., having human-like complex or hybrid N-glycan structures. In certain embodiments, N-glycans made in the host cells have a complex Gal₍₁₋₄₎GlcNAc₍₁₋₄₎Man₃GlcNAc₂ structure which may then be modified further by heterologous expression of one or more enzymes, e.g., glycosyltransferases, glycosidases such as mannosidases, sugar transporters and the like, to yield modified glycoproteins, e.g., having human-like complex or hybrid N-glycan structures. In other embodiments, N-glycans made in the host cells have a hybrid GalGlcNAcMan₅GlcNAc₂ structure which may then be modified further by heterologous expression of one or more enzymes, e.g., glycosyltransferases, glycosidases such as mannosidases, sugar transporters and the like, to yield modified glycoproteins, e.g., having human-like complex or hybrid N-glycan structures.

In one embodiment, glycoproteins made in the engineered host cells have a complex NANA₍₁₋₄₎Gal₍₁₋₄₎GlcNAc₍₁₋₄₎Man₃GlcNA₂ glycoform of N-glycan. In other embodiments, glycoproteins made in the engineered host cells have a hybrid NANAGalGlcNAcMan₅GlcNA₂ glycoform of N-glycan. Thus, in one embodiment, glycoprotein compositions of the present invention may comprise predominantly complex NANA₍₁₋₄₎Gal₍₁₋₄₎GlcNAc₍₁₋₄₎Man₃GlcNA₂ glycoforms. In other embodiments, glycoprotein compositions of the present invention will comprise predominantly hybrid NANAGalGlcNAcMan₅GlcNA₂ glycoforms. Methods for producing the Man₅GlcNAc₂ core structure and its modification to form either complex and hybrid glycoforms are provided in Gerngross, WO02/00879 and U.S. Pat. No. 7,029,872, the specification of which are hereby incorporated herein by reference for its disclosure cited herein.

Accordingly, in one embodiment, the invention provides a method for producing a human-like glycoprotein in a non-human eukaryotic host cell. The host cell of the invention is selected or engineered to be depleted in 1,6-mannosyltransferase activities which would otherwise add mannose residues onto the N-glycan on a glycoprotein. One or more enzymes (enzymatic activities) are introduced into the host cell which enable the production of a Man₅GlcNAc₂ carbohydrate structure at a high yield, e.g., at least 30 mole percent. In a preferred embodiment, at least 10% of the Man₅GlcNAc₂ produced within the host cell is a productive substrate for GnTI and thus for further glycosylation reactions in vivo and/or in vitro that produce a finished N-glycan that is similar or identical to that formed in mammals, especially humans.

In another embodiment, a nucleic acid molecule encoding one or more enzymes for production of a Man₅GlcNAc₂ carbohydrate structure is introduced into a host cell selected or engineered to be depleted in 1,6-mannosyltransferase activities. In one preferred embodiment, at least one enzyme introduced into the host cell is selected to have optimal activity at the pH of the subcellular location where the carbohydrate structure is produced. In another preferred embodiment, at least one enzyme is targeted to a host subcellular organelle where the enzyme will have optimal activity, e.g., by means of a chimeric protein comprising a cellular targeting signal peptide not normally associated with the enzyme.

The invention further provides isolated nucleic acid molecules and vectors comprising such molecules which encode an initiating α1,6-mannosyltransferase activity isolated from P. pastoris or from K. lactis. These nucleic acid molecules comprise sequences that are homologous to the OCH1 gene in S. cerevisiae. These and homologous sequences are useful for constructing host cells which will not hypermannosylate the N-glycan of a glycoprotein.

In another embodiment, the host cell is engineered to express a heterologous glycosidase, e.g., by introducing into the host one or more nucleic acid molecules encoding the glycosidase. In one embodiment, a nucleic acid molecule encodes one or more mannosidase activities involved in the production of Man₅GlcNAc₂ from Man₈GlcNAc₂ or Man₉GlcNAc₂. In a preferred embodiment, at least one of the encoded mannosidase activities has a pH optimum within 1.4 pH units of the average pH optimum of other representative enzymes in the organelle in which the mannosidase activity is localized, or has optimal activity at a pH of between about 5.1 and about 8.0, preferably between about 5.5 and about 7.5. Preferably, the heterologous enzyme is targeted to the endoplasmic reticulum, the Golgi apparatus or the transport vesicles between ER and Golgi of the host organism, where it trims N-glycans such as Man₈GlcNAc₂ to yield high levels of Man₅GlcNAc₂.

In another embodiment, the host cell is engineered to express a heterologous galactosyltransferase. In yet another embodiment, the host cell is engineered to express a heterologous sialyltransferase or a trans-sialidase.

In certain embodiments, the glycosylation enzyme is targeted to a subcellular location by forming a fusion protein between a catalytic domain of the enzyme and a cellular targeting signal peptide, e.g., by the in-frame ligation of a DNA, fragment encoding a cellular targeting signal peptide with a DNA fragment encoding a glycosylation enzyme or catalytically active fragment thereof.

In certain embodiments, the glycosylation pathway of a host is modified to express a sugar nucleotide transporter. In a preferred embodiment, a nucleotide diphosphatase enzyme is also expressed. The transporter and diphosphatase improve the efficiency of engineered glycosylation steps, by providing the appropriate substrates for the glycosylation enzymes in the appropriate compartments, reducing competitive product inhibition, and promoting the removal of nucleoside diphosphates.

In another embodiment, the host cell is engineered to express a functional CMP-sialic acid (CMP-Sia) biosynthetic pathway.

In another embodiment, a method of engineering a CMP-Sia biosynthetic pathway into a non-human eukaryotic cell is provided. The method involves the cloning and expression of several enzymes of mammalian origin, bacterial origin or both, in a host cell, particularly a fungal host cell or other host cell that lacks endogenous sialylation or that can benefit from increased levels of CMP-Sia. The engineered CMP-Sia biosynthetic pathway is useful for producing sialylated glycolipids, O-glycans and N-glycans in vivo. The present invention is thus useful for facilitating the generation of sialylated therapeutic glycoproteins in non-human host cells lacking endogenous sialylation or in non-human host cells lacking adequate levels of endogenous sialylation. Examples of non-human host cells lacking adequate levels of endogenous sialylation include lower eukaryotic host cells, insect cells and plant cells. Thus, in certain embodiments of the invention, the host cells are engineered to produce sialylated glycoproteins where none would otherwise be produced endogenously. In other embodiments, of the invention, the host cells are engineered to increase the level of sialylated glycoproteins above native endogenous levels.

The present invention also provides a combinatorial nucleic acid library useful for making fusion constructs which can target a desired protein or polypeptide fragment, e.g., an enzyme involved in glycosylation or a catalytic domain thereof, to a selected subcellular region of a host cell. In one preferred embodiment, the combinatorial nucleic acid library comprises (a) nucleic acid sequences encoding different cellular targeting signal peptides and (b) nucleic acid sequences encoding different polypeptides to be targeted. Nucleic acid sequences of or derived from (a) and (b) are ligated together to produce fusion constructs, at least one of which encodes a functional protein domain (e.g., a catalytic domain of an enzyme) ligated in-frame to a heterologous cellular targeting signal peptide, i.e., one which it normally does not associate with.

The invention also provides a method for modifying the glycosylation pathway of a host cell (e.g., any eukaryotic host cell, including a human host cell) using enzymes involved in modifying N-glycans including glycosidases and glycosyltransferases; by transforming the host cell with a nucleic acid (e.g., a combinatorial) library of the invention to produce a genetically mixed cell population expressing at least one and preferably two or more distinct chimeric glycosylation enzymes having a catalytic domain ligated in-frame to a cellular targeting signal peptide which it normally does not associate with. A host cell having a desired glycosylation phenotype may optionally be selected from the population. Host cells modified using the library and associated methods of the invention are useful, e.g., for producing glycoproteins having a glycosylation pattern similar or identical to those produced in mammals, especially humans.

In another aspect, the combinatorial library of the present invention enables production of one or a combination of catalytically active glycosylation enzymes, which successfully localize to intracellular compartments in which they function efficiently in the glycosylation/secretory pathway. Preferred enzymes convert (α-1,2-Man)₃₋₉ Man₅GlcNAc₂ to Man₅GlcNAc₂ at high efficiency in vivo. In addition, the invention provides eukaryotic host strains, and in particular, yeasts, fungal, insect, plant, plant cells, algae and insect cell hosts, capable of producing glycoprotein intermediates or products with Man₅GlcNAc₂ and/or GlcNAcMan₅GlcNAc₂ as the predominant N-glycan.

The present invention also provides methods using the combinatorial library for producing, in vivo, glycoprotein intermediates or products with predominantly Man₅GlcNAc₂ or GlcNAcMan₅GlcNAc₂ N-glycans covalently attached to proteins, e.g., recombinant proteins expressed in host cells of the invention. The present invention also provides methods for producing, in vivo, complex glycoprotein products bearing a terminal galactose, preferably having the structure Gal₍₁₋₄₎GlcNAc₍₁₋₄₎Man₃GlcNAc₂ as well as hybrid glycoprotein products bearing a terminal galactose, preferably having the structure GalGlcNAcMan₅GlcNAc₂. The present invention also provides methods for producing, in vivo, glycoprotein products bearing a terminal sialic acid, preferably having the structure NANA₍₁₋₄₎Gal₍₁₋₄₎GlcNAc₍₁₋₄₎Man₃GlcNAc₂ as well as hybrid glycoprotein products bearing a terminal sialic acid, preferably having the structure NANAGalGlcNAcMan₅GlcNAc₂.

The present invention also provides recombinant molecules derived from a combinatorial nucleic acid library; vectors, including expression vectors, comprising such recombinant molecules; proteins encoded by the recombinant molecules and vectors; host cells transformed with the recombinant molecules or vectors; and glycoproteins produced from such transformed hosts.

Further aspects of this invention include methods, compositions and kits for diagnostic and therapeutic uses in which the presence or absence on a glycoprotein of Man₅GlcNAc₂, GlcNAcMan₅GlcNAc₂, Gal₍₁₋₄₎GlcNAc₍₁₋₄₎Man₃GlcNAc₂, GalGlcNAcMan₅GlcNAc₂, NANAGalGlcNAcMan₅GlcNAc₂ and/or NANA₍₁₋₄₎Gal₍₁₋₄₎GlcNAc₍₁₋₄₎Man₃GlcNAc₂ may be detected.

In one embodiment, the invention comprises a method for producing a recombinant glycoprotein comprising sialic acid in a non-human eukaryotic host cell comprising introducing into the host cell a nucleic acid encoding a sialyltransferase enzyme. In certain embodiments, said host cell lacks an endogenous sialyltransferase activity. The sialyltransferase enzyme may be a fusion protein comprising a sialyltransferase catalytic domain and a cellular targeting signal peptide to target the sialyltransferase catalytic domain to the secretory pathway of the host cell. The cellular targeting signal peptide may be derived from Mnn2 and may comprise amino acids 1 to 108 of GenBank Accession No. (“GenBank AN”) NP_(—)009571.

The invention also comprises a method for producing a recombinant glycoprotein comprising sialic acid in a non-human eukaryotic host cell, comprising introducing into the host cell a nucleic acid encoding a trans-sialidase enzyme. In other embodiments, said nucleic acid encodes a fusion protein comprising a trans-sialidase catalytic domain with a cellular targeting signal peptide to target the trans-sialidase catalytic domain to the secretory pathway, cell wall or plasma membrane of the host cell. In one embodiment, said method further comprises supplementing the medium for growing the host cell with a sialic acid donor, such as CMP-Sia. In other embodiments, said method may comprise supplementing the medium with one or more precursors to a sialic acid donor. Such precursors may include, for example, one or more of glucosamine [GlcN], GlcNAc, UDP-GlcNAc, and ManNAc, respectively. In one embodiment, the method further comprises the step of introducing into the host cell one or more nucleic acids encoding one or more enzymes involved in the biosynthesis or transport of CMP-Sialic acid.

In one embodiment, the invention provides a method for producing a recombinant sialylated glycoprotein in a host cell, the host cell selected or engineered to produce glycoproteins comprising a Gal₍₁₋₄₎GlcNAc₍₁₋₄₎Man₃GlcNAc₂ glycoform, the method comprising the step of introducing into the host cell a nucleic acid encoding an enzyme having sialyltransferase activity, the enzyme comprising a sialyltransferase catalytic domain and a cellular targeting signal peptide to target the sialyltransferase catalytic domain to the secretory pathway of the host cell; wherein, upon passage of the recombinant glycoprotein through the secretory pathway of the host cell, a recombinant glycoprotein comprising a NANA₍₁₋₄₎Gal₍₁₋₄₎GlcNAc₍₁₋₄₎Man₃GlcNA₂ glycoform is produced. In one embodiment, the cellular targeting signal peptide targets the sialyltransferase catalytic domain to a location in the secretory pathway selected from the group consisting of the endoplasmic reticulum, the Golgi apparatus, the trans-Golgi network and secretory vesicles. In one embodiment, the method further comprises the step of introducing into the host cell a nucleic acid encoding one or more enzymes involved in the biosynthesis or transport of CMP-Sia.

In another embodiment, the invention provides a method for producing a recombinant sialylated glycoprotein in a host cell, the host cell selected or engineered to produce glycoproteins comprising a Gal₍₁₋₄₎GlcNAc₍₁₋₄₎Man₃GlcNAc₂ glycoform, the method comprising the step of introducing into the host cell: (a) a nucleic acid encoding an enzyme having sialyltransferase activity; and (b) a nucleic acid encoding one or more enzymes involved in the biosynthesis or transport of CMP-Sia; wherein upon passage of the recombinant glycoprotein through the secretory pathway of the host cell, a recombinant glycoprotein comprising a NANA₍₁₋₄₎Gal₍₁₋₄₎GlcNAc₍₁₋₄₎Man₃GlcNA₂ glycoform is produced. In one embodiment, wherein the enzyme having sialyltransferase activity comprises a sialyltransferase catalytic domain and a cellular targeting signal peptide to target the sialyltransferase catalytic domain to the secretory pathway of the host cell.

In another embodiment, the invention comprises a method for producing a recombinant sialylated glycoprotein in a host cell, the host cell selected or engineered to produce glycoproteins comprising a GalGlcNAcMan₅GlcNAc₂ glycoform, the method comprising the step of introducing into the host cell a nucleic acid encoding an enzyme having sialyltransferase activity, the enzyme comprising a sialyltransferase catalytic domain and a cellular targeting signal peptide to target the sialyltransferase catalytic domain to the secretory pathway of the host cell; wherein, upon passage of the recombinant glycoprotein through the secretory pathway of the host cell, a recombinant glycoprotein comprising a NANAGalGlcNAcMan₅GlcNA₂ glycoform is produced.

In another embodiment, the invention comprises a method for producing a recombinant sialylated glycoprotein in a host cell, the host cell selected or engineered to produce glycoproteins comprising a GalGlcNAcMan₅GlcNAc₂ glycoform, the method comprising the step of introducing into the host cell: (a) a nucleic acid encoding an enzyme having sialyltransferase activity; and (b) a nucleic acid encoding one or more enzymes involved in the biosynthesis or transport of CMP-Sia; wherein upon passage of the recombinant glycoprotein through the Golgi apparatus of the host cell, a recombinant glycoprotein comprising a NANAGalGlcNAcMan₅GlcNA₂ glycoform is produced.

The invention also comprises a method for producing a recombinant glycoprotein comprising sialic acid in a recombinant non-human eukaryotic host cell comprising introducing into medium used for growing the host cell a trans-sialidase enzyme and a sialic acid donor.

In other embodiments, the above described methods may further comprise the step of introducing into the host cell one or more additional nucleic acids encoding one or more enzymes selected from the group consisting of glycosyltransferases, glycosidases and sugar transporters.

In one embodiment, said method produces a recombinant glycoprotein comprising a NANA₍₁₋₄₎Gal₍₁₋₄₎GlcNAc₍₁₋₄₎Man₃GlcNAc₂ glycoform. In another embodiment, said method produces a recombinant glycoprotein comprising a NANAGalGlcNAcMan₅GlcNA₂ glycoform.

In certain embodiments, the host cell to be used in the claimed methods may be selected or engineered to comprise a cellular pool of CMP-sialic acid, or may be cultured in the presence of a sialic acid donor, such as CMP-sialic acid, or in the presence of a precursor of a sialic acid donor. In certain embodiments, the host cell may be selected or engineered to produce CMP-sialic acid. In other embodiments, the host cell may be modified to express one or more enzyme activities involved in the CMP-Sia pathway.

In yet other embodiments, the host cell to be used in the claimed methods may be modified to express one or more glycosylation enzymes selected from the group consisting of glycosyltransferases, glycosidases and sugar transporters. In a preferred embodiment, the host cell of the claimed methods has bee modified (selected or engineered) to express a CMP-sialic acid transporter. The glycosylation enzymes may also be fusion proteins comprising a catalytic domain and a cellular targeting signal peptide to target the catalytic domain to a subcellular location in the host cell.

The host cell to be used in the above described method may be any host cell. In some embodiments, the host cell lacks endogenous sialyltransferase activity. In some embodiments, the host cell lacks endogenous CMP-Sia. In other embodiments, the host cell is selected from the group consisting of a lower eukaryotic host cell, an insect cell or a plant cell. In other embodiments, the host cell is selected from the group consisting of any Pichia sp., any Saccharomyces sp., Hansenula polymorpha, any Kluyveromyces sp., Candida albicans, any Aspergillus sp., Trichoderma reesei, Chrysosporium lucknowense, any Fusarium sp. and Neurospora crassa.

In other embodiments, the host cell to be used in the above described methods produces a glycoprotein comprising a terminal galactose.

In some embodiments, the host cells to be used in the claimed methods may produce glycoproteins comprise a terminal galactose. In some embodiments, the host cell may produce complex glycoproteins comprising a Gal₍₁₋₄₎GlcNAc₍₁₋₄₎Man₃GlcNAc₂ glycoform. In other embodiments, the host cell may produce hybrid glycoproteins comprising a GalGlcNAcMan₅GlcNAc₂ glycoform.

In certain embodiments, the above described method may further comprise the step of introducing into the host cell which lacks endogenous CMP-Sia one or more additional nucleic acids encoding one or more enzymes involved in the biosynthesis or transport of CMP-Sialic acid. In one embodiment, the above described method comprises the step of introducing into the host cell one or more additional nucleic acids encoding one or more mammalian enzymes selected from the group consisting of: UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase, N-acetylneuraminate-9-phosphate synthase, N-acetylneuraminate-9-phosphatase, CMP-sialic acid phosphatase and CMP-sialic acid synthase. In another embodiment, the above described method comprises the step of introducing into the host cell one or more additional nucleic acids encoding one or more bacterial enzymes selected from the group consisting of: UDP-GlcNAc epimerase, sialate synthase and CMP-Sia synthase. In another embodiment, the above described method comprises the step of introducing into the host cell one or more additional nucleic acids encoding one or more bacterial enzymes selected from the group consisting of: bacterial UDP-GlcNAc epimerase (NeuC), sialate synthase (NeuB) and a mammalian CMP-Sia synthase.

The invention also comprises a nucleic acid encoding a fusion protein comprising a sialyltransferase catalytic domain and a cellular targeting signal peptide to target the sialyltransferase catalytic domain to the secretory pathway of the host cell. For example, suitable cellular targeting signal peptides may target the sialyltransferase catalytic domain to the endoplasmic reticulum, the Golgi apparatus, the trans-Golgi network, or secretory vesicles. In one embodiment, the cellular targeting signal peptide targets the sialyltransferase catalytic domain to the Golgi apparatus. In one embodiment, said cellular targeting signal comprises amino acids 1 to 108 of GenBank AN: NP_(—)00971.

The invention also comprises a nucleic acid encoding a fusion protein comprising a trans-sialidase catalytic domain and a cellular targeting signal peptide to target the trans-sialidase catalytic domain to the secretory pathway, the cell wall or cell membrane of the host cell.

The invention also comprises the host cells to be used in the methods of the invention, having the characteristics disclosed above in connection to the claimed methods. In one embodiment, a host cell of the invention comprises a non-human eukaryotic host cell that has been genetically engineered to express a nucleic acid encoding an enzyme having sialyltransferase activity or a trans-sialidase activity. In one embodiment, the host cell of the invention is a non-human eukaryotic host cell that has been genetically engineered to produce CMP-sialic acid. In one embodiment, a host cell of the invention comprises a non-human eukaryotic host cell that has been genetically engineered to express a nucleic acid encoding an enzyme having sialyltransferase activity or a trans-sialidase activity, and to produce CMP-sialic acid.

In another embodiment, the invention comprises a lower eukaryotic host cell capable of producing a recombinant glycoprotein comprising a complex NANA₍₁₋₄₎Gal₍₁₋₄₎GlcNAc₍₁₋₄₎Man₃GlcNAc₂ glycoform, or a hybrid NANAGalGlcNAcMan₅GlcNAc₂, or a NANAGalGlcNAcMan₅GlcNAc₂ glycoform.

The invention also comprises glycoprotein compositions produced according to the methods of the invention. In certain embodiments, said glycoprotein composition may predominantly comprise a complex NANA₍₁₋₄₎Gal₍₁₋₄₎GlcNAc₍₁₋₄₎Man₃GlcNAc₂ glycoform. In other embodiments, said glycoprotein composition may comprise a hybrid NANAGalGlcNAcMan₅GlcNAc₂.

The invention also comprises glycoprotein compositions produced according to the methods of the invention. In certain embodiments, said glycoprotein compositions predominantly comprise complex NANA₍₁₋₄₎Gal₍₁₋₄₎GlcNAc₍₁₋₄₎Man₃GlcNAc₂ glycoforms. In other embodiments, said glycoprotein compositions comprise predominantly hybrid NANAGalGlcNAcMan₅GlcNAc₂.

The invention also provides a method for producing a sialylated glycoprotein in a recombinant non-human eukaryotic host cell comprising introducing into medium used for growing the host a trans-sialidase enzyme and a sialic acid donor.

The invention also provides a method of enhancing the production of CMP-sialic acid in a host cell comprising the step of introducing into the host cell a nucleic acid encoding an enzyme having GlcNAc epimerase activity.

The invention also provides a method of enhancing the production of CMP-sialic acid in a host cell comprising the step of introducing into the host cell a nucleic acid encoding an enzyme having sialate aldolase activity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic diagram of a typical fungal N-glycosylation pathway.

FIG. 1B is a schematic diagram of a typical human N-glycosylation pathway.

FIG. 2 depicts construction of a combinatorial DNA library of fusion constructs. FIG. 2A diagrams the insertion of a targeting peptide fragment into pCR2.1-TOPO (Invitrogen, Carlsbad, Calif.). FIG. 2B shows the generated targeting peptide sub-library having restriction sites NotI-AscI. FIG. 2C diagrams the insertion of a catalytic domain region into pJN347, a modified pUC19 vector. FIG. 2D shows the generated catalytic domain sub-library having restriction sites NotI, AscI and PacI. FIG. 2E depicts one particular fusion construct generated from the targeting peptide sub-library and the catalytic domain sub-library.

FIG. 3 (SEQ ID NOS: 45-46 respectively, in order of appearance) illustrates the M. musculus α-1,2-mannosidase IA open reading frame. The sequences of the PCR primers used to generate N-terminal truncations are underlined.

FIGS. 4A-4F illustrates engineering of vectors with multiple auxotrophic markers and genetic integration of target proteins in the P. pastoris OCH1 locus.

FIGS. 5A-5E show MALDI-TOF analysis demonstrating production of kringle 3 domain of human plasminogen (K3) glycoproteins having Man₅GlcNAc₂ as the predominant N-glycan structure in P. pastoris. FIG. 5A depicts the standard Man₅GlcNAc₂ [a] glycan (Glyko, Novato, Calif.) and Man₅GlcNAc₂+ [b]. FIG. 5B shows PNGase-released glycans from K3 wild type. The N-glycans shown are as follows: Man₉GlcNAc₂ [d]; Man₁₀GlcNAc₂ [e]; Man₁₁GlcNAc₂ [f]; Man₁₂GlcNAc₂ [g]. FIG. 5C depicts the och1 deletion resulting in the production of Man₈GlcNAc₂ [c] as the predominant N-glycan. FIGS. 5D and 5E show the production of Man₅GlcNAc₂ [b] after in vivo trimming of Man₈GlcNAc₂ with a chimeric α-1,2-mannosidase. The predominant N-glycan is indicated by a peak with a mass (m/z) of 1253 consistent with its identification as Man₅GlcNAc₂ [b].

FIGS. 6A-6F show MALDI-TOF analysis demonstrating production of IFN-β glycoproteins having Man₅GlcNAc₂ as the predominant N-glycan structure in P. pastoris. FIG. 6A shows the standard Man₅GlcNAc₂ [a] and Man₅GlcNAc₂+Na⁺ [b] as the standard (Glyko, Novato, Calif.). FIG. 6B shows PNGase-released glycans from IFN-β wildtype. FIG. 6C depicts the och1 knock-out producing Man₈GlcNAc₂ [c]; Man₉GlcNAc₂ [d]; Man₁₀GlcNAc₂ [e]; Man₁₁GlcNAc₂ [f]; Man₁₂GlcNAc₂ [g]; and no production of Man₅GlcNAc₂ [b]. FIG. 6D shows relatively small amount of Man₅GlcNAc₂ [b] among other intermediate N-glycans Man₈GlcNAc₂ [c] to Man₁₂GlcNAc₂ [g]. FIG. 6E shows a significant amount of Man₅GlcNAc₂ [b] relative to the other glycans Man₈GlcNAc₂ [c] and Man₉GlcNAc₂ [d] produced by pGC5 (Saccharomyces MNS1 (m)/mouse mannosidase IB Δ99). FIG. 6F shows predominant production of Man₅GlcNAc₂ [b] on the secreted glycoprotein IFN-β by pFB8 (Saccharomyces SEC12 (m)/mouse mannosidase IA Δ187). The N-glycan is indicated by a peak with a mass (m/z) of 1254 consistent with its identification as Man₅GlcNAc₂ [b].

FIG. 7 shows a high performance liquid chromatogram for: (A) Man₉GlcNAc₂ standard labeled with 2-AB (negative control); (B) supernatant of medium P. pastoris, Δoch1 transformed with pFB8 mannosidase, which demonstrates a lack of extracellular mannosidase activity in the supernatant; and (C) Man₉GlcNAc₂ standard labeled with 2-AB after exposure to T. reesei mannosidase (positive control).

FIG. 8 shows a high performance liquid chromatogram for: (A) Man₉GlcNAc₂ standard labeled with 2-AB (negative control); (B) supernatant of medium P. pastoris, Δoch1 transformed with pGC5 mannosidase, which demonstrates a lack of extracellular mannosidase activity in the supernatant; and (C) Man₉GlcNAc₂ standard labeled with 2-AB after exposure to T. reesei mannosidase (positive control).

FIG. 9 shows a high performance liquid chromatogram for: (A) Man₉GlcNAc₂ standard labeled with 2-AB (negative control); (B) supernatant of medium P. pastoris, Δoch1 transformed with pBC18-5 mannosidase, which demonstrates lack of extracellular mannosidase activity in the supernatant; and (C) supernatant of medium P. pastoris, Δoch1 transformed with pDD28-3, which demonstrates activity in the supernatant (positive control).

FIGS. 10A-10B demonstrate the activity of an UDP-GlcNAc transporter in the production of GlcNAcMan₅GlcNAc₂ in P. pastoris. FIG. 10A depicts a P. pastoris strain (YSH-3) with a human GnTI but without the UDP-GlcNAc transporter resulting in some production of GlcNAcMan₅GlcNAc₂ [b] but a predominant production of Man₅GlcNAc₂ [a]. FIG. 10B depicts the addition of UDP-GlcNAc transporter from K. lactis in a strain (PBP-3) with the human GnTI, which resulted in the predominant production of GlcNAcMan₅GlcNAc₂ [b]. The single prominent peak of mass (m/z) at 1457 is consistent with its identification as GlcNAcMan₅GlcNAc₂ [b] as shown in FIG. 10B.

FIG. 11 shows a pH optimum of a heterologous mannosidase enzyme encoded by pBB27-2 (Saccharomyces MNN10 (s)/C. elegans mannosidase IB Δ31) expressed in P. pastoris.

FIGS. 12A-12C show MALDI-TOF analysis of N-glycans released from a cell free extract of K. lactis. FIG. 12A shows the N-glycans released from wild-type cells, which includes high-mannose type N-glycans. FIG. 12B shows the N-glycans released from och1 mnn1 deleted cells, revealing a distinct peak of mass (m/z) at 1908 consistent with its identification as Man₉GlcNAc₂ [d]. FIG. 12C shows the N-glycans released from och1 mnn1 deleted cells after in vitro α-1,2-mannosidase digest corresponding to a peak consistent with Man₅GlcNAc₂.

FIG. 13 represents T-DNA cassettes with catalytic domain(s) of glycosylation enzymes fused in-frame to different leader sequences. The ends of the T-DNA are marked by the right (rb) and left borders (lb). Various promoters and terminators may also be used. The plant selectable marker can also be varied. The right and left borders are required only for agrobacterium-mediated transformation and not for particle bombardment or electroporation.

FIG. 14 illustrates the CMP-sialic acid biosynthetic pathway in mammals and bacteria. Enzymes involved in each pathway are italicized. The primary substrates, intermediates and products are in bold. (PEP: phosphoenol pyruvate; CTP: cytidine triphosphate).

FIG. 15 shows the open reading frame (ORF) of E. coli protein NeuC (Genbank AN: M84026.1; SEQ ID NO: 57) and the predicted amino acid sequence (SEQ ID NO:58). The underlined DNA sequences are regions to which primers have been designed to amplify the ORF.

FIG. 16 shows the ORF of E. coli protein NeuB (Genbank AN: U05248.1; SEQ ID NO:59) and the predicted amino acid sequence (SEQ ID NO:60). The underlined DNA sequences are regions to which primers have been designed to amplify the ORF.

FIG. 17 shows the ORF of E. coli protein NeuA (Genbank AN: J05023.1; SEQ ID NO:61) and the predicted amino acid sequence (SEQ ID NO:62). The underlined DNA sequences are regions to which primers have been designed to amplify the ORF.

FIG. 18 shows the ORF of Mus musculus CMP-Sia synthase (Genbank AN: AJ006215; SEQ ID NO:63) and the amino acid sequence (SEQ ID NO:64). The underlined DNA sequences are regions to which primers have been designed to amplify the ORF.

FIG. 19 illustrates an alternative biosynthetic route for generating N-acetylmannosamine (ManNAc) in vivo. Enzymes involved in each pathway are italicized. The primary substrates, intermediates and products are in bold.

FIG. 20 shows the ORF of Sus scrofa GlcNAc epimerase (Genbank AN: D83766; SEQ ID NO:65) and the amino acid sequence (SEQ ID NO:66). The underlined DNA sequences are regions to which primers have been designed to amplify the ORF.

FIG. 21 illustrates the reversible reaction catalyzed by sialate aldolase and its dependence on sialic acid (Sia) concentration. Enzymes involved in each pathway are italicized. The primary substrates, intermediates and products are in bold.

FIG. 22 shows the ORF of E. coli sialate aldolase (Genbank AN: X03345; SEQ ID NO:67) and the amino acid sequence (SEQ ID NO:68). The underlined DNA sequences are regions to which primers have been designed to amplify the ORF.

FIG. 23 shows a HPLC of negative control of cell extracts from strain YSH99a incubated under assay conditions (Example 16) in the absence of acceptor glycan. The doublet peak eluting at 26.5 min results from contaminating cellular component(s).

FIG. 24 shows a HPLC of positive control cell extract from strain YSH99a incubated under assay conditions (Example 16) in the presence of 2-AB (aminobenzamide) labeled acceptor glycan and supplemented with CMP-sialic acid. The peak eluting at 23 min corresponds to sialylation on each branch of a biantennary galactosylated N-glycan. The doublet peak eluting at 26.5 min results from contaminating cellular component(s).

FIG. 25 shows a HPLC of a cell extract from strain YSH99a incubated under assay conditions (Example 16) in the presence of acceptor glycan with no exogenous CMP-sialic acid. The peaks eluting at 20 and 23 min correspond to mono- and di-sialylation of a biantennary galactosylated N-glycan. The doublet peak eluting at 26.5 min results from contaminating cellular component(s).

FIG. 26 shows sialidase treatment of N-glycans from YSH99a extract incubation. The sample illustrated in FIG. 25 was incubated overnight at 37° C. in the presence of 100 U sialidase (New England Biolabs, Beverley, Mass.). The peaks eluting at 20 and 23 min corresponding to mono- and di-sialylated N-glycan, have been removed in FIG. 25. The contaminating peak at 26 min remains.

FIG. 27 shows commercial mono- and di-sialylated N-glycan standards. The peaks eluting at 20 and 23 min correspond to mono- and di-sialylation of the commercial standards A1 and A2 (Glyko Inc., San Rafael, Calif.).

FIG. 28 illustrates vector pSH321b used in Example 17.

FIG. 29 depicts vector pJN711b used in Example 17.

FIG. 30 depicts vector pSH326b used in Example 17.

FIG. 31 depicts vector pSH370 used in Example 17.

FIG. 32 depicts vector pSH373 used in Example 17.

FIG. 33 shows the nucleic acid (SEQ ID NO:101) and amino acid sequence (SEQ ID NO:102) of the codon-optimized hST6Gal leader 53 fusion described in Example 17.

FIG. 34 depicts vector pSH568 used in Example 17.

FIG. 35 depicts MALDI-TOF MS analysis of the glycans produced in YSH126 and YSH145. The “+ve” sign at the upper right corner of each MALDI-ROF indicates analysis of positive ions and the “−ve” sign indicates analysis of negative ions.

FIG. 36 shows MALDI-TOF MS analysis of the glycans produced in YSH145 and YSH160.

FIG. 37 shows MALDI-TOF MS analysis of the glycans produced in YSH272. The solid arrows represent sialic acid containing glycans. The dashed arrows represent neutral glycans.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The methods and techniques of the present invention are generally performed according to conventional methods well known in the art. Generally, nomenclatures used in connection with, and techniques of biochemistry, enzymology, molecular and cellular biology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art.

The methods and techniques of the present invention are generally performed according to conventional methods well-known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992, and Supplements to 2002); Harlow and Lane Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990); Introduction to Glycobiology, Maureen E. Taylor, Kurt Drickamer, Oxford Univ. Press (2003); Worthington Enzyme Manual, Worthington Biochemical Corp. Freehold, N.J.; Handbook of Biochemistry: Section A Proteins Vol I 11976 CRC Press; Handbook of Biochemistry: Section A Proteins Vol II 1976 CRC Press; Essentials of Glycobiology, Cold Spring Harbor Laboratory Press (1999). The nomenclatures used in connection with, and the laboratory procedures and techniques of, molecular and cellular biology, protein biochemistry, enzymology and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art.

All publications, patents and other references mentioned herein are incorporated by reference.

The following terms, unless otherwise indicated, shall be understood to have the following meanings:

As used herein, the term “N-glycan” refers to an N-linked oligosaccharide, e.g., one that is attached by an asparagine-N-acetylglucosamine linkage to an asparagine residue of a polypeptide. N-glycans have a common pentasaccharide core of Man₃GlcNAc₂ (“Man” refers to mannose; “Glc” refers to glucose; and “NAc” refers to N-acetyl; GlcNAc refers to N-acetylglucosamine). The term “trimannose core” used with respect to the N-glycan also refers to the structure Man₃GlcNAc₂ (“Man₃”). N-glycans differ with respect to the number of branches (antennae) comprising peripheral sugars (e.g., fucose and sialic acid) that are added to the Man₃ core structure. N-glycans are classified according to their branched constituents (e.g., high mannose, complex or hybrid).

A “high mannose” type N-glycan has five or more mannose residues. A “complex” type N-glycan typically has at least one GlcNAc attached to the 1,3 mannose arm and at least one GlcNAc attached to the 1,6 mannose arm of the trimannose core. Complex N-glycans may also have galactose (“Gal”) residues that are optionally modified with sialic acid or derivatives (“NeuAc”, where “Neu” refers to neuraminic acid and “Ac” refers to acetyl). A complex N-glycan typically has at least one branch that terminates in an oligosaccharide such as, for example: NeuNAc-; NeuAca2-6GalNAca1-; NeuAca2-3Galb1-3GalNAca1-; NeuAca2-3/6Galb1-4GlcNAcb1-; GlcNAca1-4Galb1-(mucins only); Fuca1-2Galb1-(blood group H). Sulfate esters can occur on galactose, GalNAc, and GlcNAc residues, and phosphate esters can occur on mannose residues. NeuAc (Neu: neuraminic acid; Ac:acetyl) can be O-acetylated or replaced by NeuGl (N-glycolylneuraminic acid). Complex N-glycans may also have intrachain substitutions comprising “bisecting” GlcNAc and core fucose (“Fuc”). A “hybrid” N-glycan has at least one GlcNAc on the terminal of the 1,3 mannose arm of the trimannose core and zero or more mannoses on the 1,6 mannose arm of the trimannose core.

The term “homologs” used with respect to an original enzyme or gene of a first family or species refers to distinct enzymes or genes of a second family or species which are determined by functional, structural or genomic analyses to be an enzyme or gene of the second family or species which corresponds to the original enzyme or gene of the first family or species. Most often, homologs will have functional, structural or genomic similarities. Techniques are known by which homologs of an enzyme or gene can readily be cloned using genetic probes and PCR. Identity of cloned sequences as homolog can be confirmed using functional assays and/or by genomic mapping of the genes.

The term “predominant” or “predominantly” used with respect to the production of N-glycans refers to a structure which represents the major peak detected by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF) analysis.

Abbreviations used herein are of common usage in the art, see, e.g., abbreviations of sugars, above. Other common abbreviations include “PNGase”, which refers to peptide N-glycosidase F (EC 3.2.2.18); “GlcNAc Tr” or “GnT,” which refers to N-acetylglucosaminyl Transferase enzymes; “NANA” refers to N-acetylneuraminic acid.

As used herein, a “humanized glycoprotein” or a “human-like glycoprotein” refers alternatively to a protein having attached thereto N-glycans having less than four mannose residues, and synthetic glycoprotein intermediates (which are also useful and can be manipulated further in vitro or in vivo) having at least five mannose residues. Preferably, glycoproteins produced according to the invention contain at least 30 mole %, preferably at least 40 mole % and more preferably 50-100 mole % of the Man₅GlcNAc₂ intermediate, at least transiently. This may be achieved, e.g., by engineering a host cell of the invention to express a “better”, i.e., a more efficient glycosylation enzyme. For example, a mannosidase is selected such that it will have optimal activity under the conditions present at the site in the host cell where proteins are glycosylated and is introduced into the host cell preferably by targeting the enzyme to a host cell organelle where activity is desired.

The term “enzyme”, when used herein in connection with altering host cell glycosylation, refers to a molecule having at least one enzymatic activity, and includes full-length enzymes, catalytically active fragments, chimerics, complexes, and the like. A “catalytically active fragment” of an enzyme refers to a polypeptide having a detectable level of functional (enzymatic) activity.

A “lower eukaryotic host cell,” when used herein in connection with glycosylation profiles, refers to any eukaryotic cell which ordinarily produces high mannose containing N-glycans, and thus is meant to include, in a functional sense, some animal or plant cells whose glycosylation profiles are like those of lower eukaryotic cells, and most typical lower eukaryotic cells, including uni- and multicellular fungal and algal cells.

As used herein, the term “secretion pathway” refers to the assembly line of various glycosylation enzymes to which a lipid-linked oligosaccharide precursor and an N-glycan substrate are sequentially exposed, following the molecular flow of a nascent polypeptide chain from the cytoplasm to the endoplasmic reticulum (ER) and the compartments of the Golgi apparatus. The term “secretory pathway” thus refers to organelles and components within the cell where glycoproteins are modified in preparation for secretion. The secretory pathway includes the endoplasmic reticulum or ER, the Golgi apparatus, the trans-Golgi network and the secretory vesicles. Enzymes are said to be localized along this pathway. An enzyme X that acts on a lipid-linked glycan or an N-glycan before enzyme Y is said to be or to act “upstream” to enzyme Y; similarly, enzyme Y is or acts “downstream” from enzyme X.

The term “targeting peptide” a “targeting signal peptide” as used herein refers to nucleotide or amino acid sequences encoding a cellular targeting signal peptide which mediates the localization (or retention) of an associated sequence to sub-cellular locations, e.g., organelles.

The term “polynucleotide” or “nucleic acid molecule” refers to a polymeric form of nucleotides of at least 10 bases in length. The term includes DNA molecules (e.g., cDNA or genomic or synthetic DNA) and RNA molecules (e.g., mRNA or synthetic RNA), as well as analogs of DNA or RNA containing non-natural nucleotide analogs, non-native internucleoside bonds, or both. The nucleic acid can be in any topological conformation. For instance, the nucleic acid can be single-stranded, double-stranded, triple-stranded, quadruplexed, partially double-stranded, branched, hairpinned, circular, or in a padlocked conformation. The term includes single and double stranded forms of DNA. A nucleic acid molecule of this invention may include both sense and antisense strands of RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of the above. They may be modified chemically or biochemically or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those of skill in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids, etc.) Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions. Such molecules are known in the art and include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of the molecule.

Unless otherwise indicated, a “nucleic acid comprising SEQ ID NO:X” refers to a nucleic acid, at least a portion of which has either (i) the sequence of SEQ ID NO:X, or (ii) a sequence complementary to SEQ ID NO:X. The choice between the two is dictated by the context. For instance, if the nucleic acid is used as a probe, the choice between the two is dictated by the requirement that the probe be complementary to the desired target.

An “isolated” or “substantially pure” nucleic acid or polynucleotide (e.g., an RNA, DNA or a mixed polymer) is one which is substantially separated from other cellular components that naturally accompany the native polynucleotide in its natural host cell, e.g., ribosomes, polymerases, and genomic sequences with which it is naturally associated. The term embraces a nucleic acid or polynucleotide that (1) has been removed from its naturally occurring environment, (2) is not associated with all or a portion of a polynucleotide in which the “isolated polynucleotide” is found in nature, (3) is operatively linked to a polynucleotide which it is not linked to in nature, or (4) does not occur in nature. The term “isolated” or “substantially pure” also can be used in reference to recombinant or cloned DNA isolates, chemically synthesized polynucleotide analogs, or polynucleotide analogs that are biologically synthesized by heterologous systems.

However, “isolated” does not necessarily require that the nucleic acid or polynucleotide so described has itself been physically removed from its native environment. For instance, an endogenous nucleic acid sequence in the genome of an organism is deemed “isolated” herein if a heterologous sequence (i.e., a sequence that is not naturally adjacent to this endogenous nucleic acid sequence) is placed adjacent to the endogenous nucleic acid sequence, such that the expression of this endogenous nucleic acid sequence is altered. By way of example, a non-native promoter sequence can be substituted (e.g., by homologous recombination) for the native promoter of a gene in the genome of a human cell, such that this gene has an altered expression pattern. This gene would now become “isolated” because it is separated from at least some of the sequences that naturally flank it.

A nucleic acid is also considered “isolated” if it contains any modifications that do not naturally occur to the corresponding nucleic acid in a genome. For instance, an endogenous coding sequence is considered “isolated” if it contains an insertion, deletion or a point mutation introduced artificially, e.g., by human intervention. An “isolated nucleic acid” also includes a nucleic acid integrated into a host cell chromosome at a heterologous site, a nucleic acid construct present as an episome. Moreover, an “isolated nucleic acid” can be substantially free of other cellular material, or substantially free of culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.

As used herein, the phrase “degenerate variant” of a reference nucleic acid sequence encompasses nucleic acid sequences that can be translated, according to the standard genetic code, to provide an amino acid sequence identical to that translated from the reference nucleic acid sequence.

The term “percent sequence identity” or “identical” in the context of nucleic acid sequences refers to the residues in the two sequences which are the same when aligned for maximum correspondence. The length of sequence identity comparison may be over a stretch of at least about nine nucleotides, usually at least about 20 nucleotides, more usually at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 32 nucleotides, and preferably at least about 36 or more nucleotides. There are a number of different algorithms known in the art which can be used to measure nucleotide sequence identity. For instance, polynucleotide sequences can be compared using FASTA, Gap or Bestfit, which are programs in Wisconsin Package Version 10.0, Genetics Computer Group (GCG), Madison, Wis. FASTA provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson, 1990, hereby incorporated herein by reference). For instance, percent sequence identity between nucleic acid sequences can be determined using FASTA with its default parameters (a word size of 6 and the NOPAM factor for the scoring matrix) or using Gap with its default parameters as provided in GCG Version 6.1, hereby incorporated herein by reference.

The terms “significant alignment”, “substantial homology” or “substantial similarity,” when referring to a nucleic acid or fragment thereof, indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 50%, more preferably 60% of the nucleotide bases, usually at least about 70%, more usually at least about 80%, preferably at least about 90%, and more preferably at least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST or Gap, as discussed above.

Alternatively, significant alignment, substantial homology or similarity exists when a nucleic acid or fragment thereof hybridizes to another nucleic acid, to a strand of another nucleic acid, or to the complementary strand thereof, under stringent hybridization conditions. “Stringent hybridization conditions” and “stringent wash conditions” in the context of nucleic acid hybridization experiments depend upon a number of different physical parameters. Nucleic acid hybridization will be affected by such conditions as salt concentration, temperature, solvents, the base composition of the hybridizing species, length of the complementary regions, and the number of nucleotide base mismatches between the hybridizing nucleic acids, as will be readily appreciated by those skilled in the art. One having ordinary skill in the art knows how to vary these parameters to achieve a particular stringency of hybridization.

In general, “stringent hybridization” is performed at about 25° C. below the thermal melting point (T_(m)) for the specific DNA hybrid under a particular set of conditions. “Stringent washing” is performed at temperatures about 5° C. lower than the T_(m) for the specific DNA hybrid under a particular set of conditions. The T_(m) is the temperature at which 50% of the target sequence hybridizes to a perfectly matched probe. See Sambrook et al., supra, page 9.51, hereby incorporated herein by reference. For purposes herein, “high stringency conditions” are defined for solution phase hybridization as aqueous hybridization (i.e., free of formamide) in 6×SSC (where 20×SSC contains 3.0 M NaCl and 0.3 M sodium citrate), 1% SDS at 65° C. for 8-12 hours, followed by two washes in 0.2×SSC, 0.1% SDS at 65° C. for 20 minutes. It will be appreciated by the skilled artisan that hybridization at 65° C. will occur at different rates depending on a number of factors including the length and percent identity of the sequences which are hybridizing.

The nucleic acids (also referred to as polynucleotides) of this invention may include both sense and antisense strands of RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of the above. They may be modified chemically or biochemically or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those of skill in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids, etc.). Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions. Such molecules are known in the art and include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of the molecule.

The term “mutated” when applied to nucleic acid sequences means that nucleotides in a nucleic acid sequence may be inserted, deleted or changed compared to a reference nucleic acid sequence. A single alteration may be made at a locus (a point mutation) or multiple nucleotides may be inserted, deleted or changed at a single locus. In addition, one or more alterations may be made at any number of loci within a nucleic acid sequence. A nucleic acid sequence may be mutated by any method known in the art including but not limited to mutagenesis techniques such as “error-prone PCR” (a process for performing PCR under conditions where the copying fidelity of the DNA polymerase is low, such that a high rate of point mutations is obtained along the entire length of the PCR product. See, e.g., Leung, 1989; Caldwell, 1992; and “oligonucleotide-directed mutagenesis” (a process which enables the generation of site-specific mutations in any cloned DNA segment of interest. See, e.g., Reidhaar-Olson 1988).

The term “vector” as used herein is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Other vectors include cosmids, bacterial artificial chromosomes (BAC) and yeast artificial chromosomes (YAC). Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome (discussed in more detail below). Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., vectors having an origin of replication which functions in the host cell). Other vectors can be integrated into the genome of a host cell upon introduction into the host cell, and are thereby replicated along with the host genome. Moreover, certain preferred vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”).

“Operatively linked” expression control sequences refers to a linkage in which the expression control sequence is contiguous with the gene of interest to control the gene of interest, as well as expression control sequences that act in trans or at a distance to control the gene of interest.

The term “expression control sequence” as used herein refers to polynucleotide sequences which are necessary to affect the expression of coding sequences to which they are operatively linked. Expression control sequences are sequences which control the transcription, post-transcriptional events and translation of nucleic acid sequences. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., ribosome binding sites); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence. The term “control sequences” is intended to include, at a minimum, all components whose presence is essential for expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.

The term “recombinant host cell” (or simply “host cell”), as used herein, is intended to refer to a cell that has been genetically engineered. A recombinant host cell includes a cell into which a recombinant vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein. A recombinant host cell may be an isolated cell or cell line grown in culture or may be a cell which resides in a living tissue or organism. The term “host” refers to any organism, animal or plant, comprising one or more “host cells”, or to the source of the “host cells”.

Moreover, as used herein a “host cell which lacks endogenous CMP-Sia” refers to a cell that does not endogenously produce CMP-Sia, including cells which lack a CMP-Sia pathway. As used herein, a “fungal host cell” refers to a fungal host cell that lacks CMP-Sia.

The term “peptide” as used herein refers to a short polypeptide, e.g., one that is typically less than about 50 amino acids long and more typically less than about 30 amino acids long. The term as used herein encompasses analogs and mimetics that mimic structural and thus biological function.

The term “polypeptide” as used herein encompasses both naturally-occurring and non-naturally-occurring proteins, and fragments, mutants, derivatives and analogs thereof. A polypeptide may be monomeric or polymeric. Further, a polypeptide may comprise a number of different domains each of which has one or more distinct activities.

The term “isolated protein” or “isolated polypeptide” is a protein or polypeptide that by virtue of its origin or source of derivation (1) is not associated with naturally associated components that accompany it in its native state, (2) when it exists in a purity not found in nature, where purity can be adjudged with respect to the presence of other cellular material (e.g., is free of other proteins from the same species) (3) is expressed by a cell from a different species, or (4) does not occur in nature (e.g., it is a fragment of a polypeptide found in nature or it includes amino acid analogs or derivatives not found in nature or linkages other than standard peptide bonds). Thus, a polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be “isolated” from its naturally associated components. A polypeptide or protein may also be rendered substantially free of naturally associated components by isolation, using protein purification techniques well-known in the art. As thus defined, “isolated” does not necessarily require that the protein, polypeptide, peptide or oligopeptide so described has been physically removed from its native environment.

The term “polypeptide fragment” as used herein refers to a polypeptide that has an amino-terminal and/or carboxy-terminal deletion compared to a full-length polypeptide. In a preferred embodiment, the polypeptide fragment is a contiguous sequence in which the amino acid sequence of the fragment is identical to the corresponding positions in the naturally-occurring sequence. Fragments typically are at least 5, 6, 7, 8, 9 or 10 amino acids long, preferably at least 12, 14, 16 or 18 amino acids long, more preferably at least 20 amino acids long, more preferably at least 25, 30, 35, 40 or 45, amino acids, even more preferably at least 50 or 60 amino acids long, and even more preferably at least 70 amino acids long.

A “modified derivative” refers to polypeptides or fragments thereof that are substantially homologous in primary structural sequence but which include, e.g., in vivo or in vitro chemical and biochemical modifications or which incorporate amino acids that are not found in the native polypeptide. Such modifications include, for example, acetylation, carboxylation, phosphorylation, glycosylation, ubiquitination, labeling, e.g., with radionuclides, and various enzymatic modifications, as will be readily appreciated by those well skilled in the art. A variety of methods for labeling polypeptides and of substituents or labels useful for such purposes are well-known in the art, and include radioactive isotopes such as ¹²⁵I, ³²P, ³⁵S, and ³H, ligands which bind to labeled antiligands (e.g., antibodies), fluorophores, chemiluminescent agents, enzymes, and antiligands which can serve as specific binding pair members for a labeled ligand. The choice of label depends on the sensitivity required, ease of conjugation with the primer, stability requirements, and available instrumentation. Methods for labeling polypeptides are well-known in the art. See Ausubel et al., 1992, hereby incorporated by reference.

A “polypeptide mutant” or “mutein” or “variant” refers to a polypeptide whose sequence contains an insertion, duplication, deletion, rearrangement or substitution of one or more amino acids compared to the amino acid sequence of a native or wild type protein. A mutein may have one or more amino acid point substitutions, in which a single amino acid at a position has been changed to another amino acid, one or more insertions and/or deletions, in which one or more amino acids are inserted or deleted, respectively, in the sequence of the naturally-occurring protein, and/or truncations of the amino acid sequence at either or both the amino or carboxy termini. A mutein may have the same but preferably has a different biological activity compared to the naturally-occurring protein.

A mutein has at least 70% overall sequence homology to its wild-type counterpart. Even more preferred are muteins having 80%, 85% or 90% overall sequence homology to the wild-type protein. In an even more preferred embodiment, a mutein exhibits 95% sequence identity, even more preferably 97%, even more preferably 98% and even more preferably 99% overall sequence identity. Sequence homology may be measured by any common sequence analysis algorithm, such as Gap or Bestfit.

Preferred amino acid substitutions are those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinity or enzymatic activity, and (5) confer or modify other physicochemical or functional properties of such analogs.

As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. See Immunology—A Synthesis (2^(nd) Edition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. Stereoisomers (e.g., D-amino acids) of the twenty conventional amino acids, unnatural amino acids such as α-, α-disubstituted amino acids, N-alkyl amino acids, and other unconventional amino acids may also be suitable components for polypeptides of the present invention. Examples of unconventional amino acids include: 4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, s-N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, the left-hand direction is the amino terminal direction and the right hand direction is the carboxy-terminal direction, in accordance with standard usage and convention.

A protein has “homology” or is “homologous” to a second protein if the nucleic acid sequence that encodes the protein has a similar sequence to the nucleic acid sequence that encodes the second protein. Alternatively, a protein has homology to a second protein if the two proteins have “similar” amino acid sequences. (Thus, the term “homologous proteins” is defined to mean that the two proteins have similar amino acid sequences). In a preferred embodiment, a homologous protein is one that exhibits 60% sequence homology to the wild type protein, more preferred is 70% sequence homology. Even more preferred are homologous proteins that exhibit 80%, 85% or 90% sequence homology to the wild type protein. In a yet more preferred embodiment, a homologous protein exhibits 95%, 97%, 98% or 99% sequence identity. As used herein, homology between two regions of amino acid sequence (especially with respect to predicted structural similarities) is interpreted as implying similarity in function.

When “homologous” is used in reference to proteins or peptides, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions. A “conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of homology may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art (see, e.g., Pearson et al., 1994, hereby incorporated herein by reference).

The following six groups each contain amino acids that are conservative substitutions for one another: 1) Serine (S), Threonine (T); 2) Aspartic Acid (D), Glutamic Acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Alanine (A), Valine (V), and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

Sequence homology for polypeptides, which is also referred to as percent sequence identity, is typically measured using sequence analysis software. See, e.g., the Sequence Analysis Software Package of the Genetics Computer Group (GCG), University of Wisconsin Biotechnology Center, 910 University Avenue, Madison, Wis. 53705. Protein analysis software matches similar sequences using measure of homology assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, GCG contains programs such as “Gap” and “Bestfit” which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutein thereof. See, e.g., GCG Version 6.1.

A preferred algorithm when comparing a inhibitory molecule sequence to a database containing a large number of sequences from different organisms is the computer program BLAST (Altschul, 1990; Gish, 1993; Madden, 1996; Altschul, 1997; Zhang, 1997), especially blastp or tblastn (Altschul, 1997). Preferred parameters for BLASTp are: Expectation value: 10 (default); Filter: seg (default); Cost to open a gap: 11 (default); Cost to extend a gap: 1 (default); Max. alignments: 100 (default); Word size: 11 (default); No. of descriptions: 100 (default); Penalty Matrix: BLOWSUM62.

The length of polypeptide sequences compared for homology will generally be at least about 16 amino acid residues, usually at least about 20 residues, more usually at least about 24 residues, typically at least about 28 residues, and preferably more than about 35 residues. When searching a database containing sequences from a large number of different organisms, it is preferable to compare amino acid sequences. Database searching using amino acid sequences can be measured by algorithms other than blastp known in the art. For instance, polypeptide sequences can be compared using FASTA, a program in GCG Version 6.1. FASTA provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson, 1990, hereby incorporated herein by reference). For example, percent sequence identity between amino acid sequences can be determined using FASTA with its default parameters (a word size of 2 and the PAM250 scoring matrix), as provided in GCG Version 6.1, hereby incorporated herein by reference.

The term “fusion protein” refers to a polypeptide comprising a polypeptide or fragment coupled to heterologous amino acid sequences. Fusion proteins are useful because they can be constructed to contain two or more desired functional elements from two or more different proteins. A fusion protein comprises at least 10 contiguous amino acids from a polypeptide of interest, more preferably at least 20 or 30 amino acids, even more preferably at least 40, 50 or 60 amino acids, yet more preferably at least 75, 100 or 125 amino acids. Fusion proteins can be produced recombinantly by constructing a nucleic acid sequence which encodes the polypeptide or a fragment thereof in-frame with a nucleic acid sequence encoding a different protein or peptide and then expressing the fusion protein. Alternatively, a fusion protein can be produced chemically by crosslinking the polypeptide or a fragment thereof to another protein.

The term “region” as used herein refers to a physically contiguous portion of the primary structure of a biomolecule. In the case of proteins, a region is defined by a contiguous portion of the amino acid sequence of that protein.

The term “domain” as used herein refers to a structure of a biomolecule that contributes to a known or suspected function of the biomolecule. Domains may be co-extensive with regions or portions thereof; domains may also include distinct, non-contiguous regions of a biomolecule. Examples of protein domains include, but are not limited to, an Ig domain, an extracellular domain, a transmembrane domain, and a cytoplasmic domain.

As used herein, the term “molecule” means any compound, including, but not limited to, a small molecule, peptide, protein, sugar, nucleotide, nucleic acid, lipid, etc., and such a compound can be natural or synthetic.

“Specific binding” refers to the ability of two molecules to bind to each other in preference to binding to other molecules in the environment. Typically, “specific binding” discriminates over adventitious binding in a reaction by at least two-fold, more typically by at least 10-fold, often at least 100-fold. Typically, the affinity or avidity of a specific binding reaction is at least about 10⁻⁷ M (e.g., at least about 10⁻⁸ M or 10⁻⁹ M).

As used herein, a “CMP-Sialic acid biosynthetic pathway” or a “CMP-Sia biosynthetic pathway” refers to one or more glycosylation enzymes which results in the formation of CMP-Sia in a host.

As used herein, a “CMP-Sia pool” refers to a detectable level of cellular CMP-Sia. The CMP-Sia pool may be the result of the production of CMP-Sia by the host cell, or of the uptake of CMP-Sia from the culture media.

The substrate UDP-GlcNAc is the abbreviation for UDP-N-acetylglucosamine. The intermediate ManNAc is the abbreviation for N-acetylmannosamine. The intermediate ManNAc-6-P is the abbreviation for N-acetylmannosamine-6-phosphate. The intermediate Sia-9-P is the abbreviation for sialate-9-phosphate. The intermediate Cytidine monophosphate-sialic acid is abbreviated as “CMP-Sia.” Sialic acid is abbreviated as “Sia,” “Neu5Ac,” “NeuAc” or “NANA” herein.

As used herein, the term “sialic acid” refers to a group of molecules where the common molecule includes N-acetyl-5-neuraminic acid (Neu5Ac) having the basic 9-carbon neuraminic acid core modified at the 5-carbon position with an attached acetyl group. Common derivatives of Neu5Ac at the 5-carbon position include: 2-keto-3-deoxy-d-glycero-d-galactonononic acid (KDN) which possesses a hydroxyl group in place of the acetyl group; de-N-acetylation of the 5-N-acetyl group produces neuraminic (Neu); hydroxylation of the 5-N-acetyl group produces N-glycolylneuraminic acid (Neu5Gc). The hydroxyl groups at positions 4-, 7-, 8- and 9- of these four molecules (Neu5Ac, KDN, Neu and Neu5Gc) can be further substituted with O-acetyl, O-methyl, O-sulfate and phosphate groups to enlarge this group of compounds. Furthermore, unsaturated and dehydro forms of sialic acids are known to exist.

The gene encoding for the UDP-GlcNAc epimerase is abbreviated as “NeuC.” The gene encoding for the sialate synthase is abbreviated as “NeuB.” The gene encoding for the CMP-Sialate synthase is abbreviated as “NeuA.”

Sialate aldolase is also commonly referred to as sialate lyase and sialate pyruvate-lyase. More specifically in E. coli, sialate aldolase is referred to as NanA. See Ringerberg et al. (2001).

As used herein, the term “sialic acid donor” refers to a molecule or entity that is capable of providing a sialic acid residue for transfer to a substrate by enzymatic or synthetic means. A sialic acid donor can be a sugar nucleotide precursor such as CMP-Sia. In certain embodiments of the invention, a precursor to a sialic acid donor may be used, in conjunction with adapted host cells which are competent for conversion of the precursor molecule to the sialic acid donor. Such adapted host cells may, for example have be capable of producing one or more sugar nucleotide precursors of CMP-Sia such as UDP-GlcNAc [glucosamine] and ManNAc. Thus the “sialic acid donor” may be provided by providing an adapted host cell which contains one or more sugar nucleotide precursors, such as UDP-GlcNAc or ManNAc, and which has functional enzyme activity for the conversion of these molecules to CMP-Sialic acid. The host cell may have such enzyme activity endogenously, or may be engineered to express enzymes which are able to convert UDP-GlcNAc or ManNAc to CMP-Sia. In other embodiments, the host cell may be cultured in media containing the necessary enzymes for the conversion of the precursor molecule to the sialic acid donor.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice of the present invention and will be apparent to those of skill in the art. All publications and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. The materials, methods, and examples are illustrative only and not intended to be limiting. Throughout this specification and claims, the word “comprise” or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

Methods for Producing Host Cells Having Man₅GlcNAc₂ Modified Oligosaccharides for the Generation of Human-Like N-Glycans

The invention provides a method for producing a glycoprotein having human-like glycosylation in a non-human eukaryotic host cell. As described in more detail below, a eukaryotic host cell that does not naturally express, or which is engineered not to express, one or more enzymes involved in production of high mannose structures is selected as a starting host cell. Such a selected host cell is engineered to express one or more enzymes or other factors required to produce human-like glycoproteins. A desired host strain can be engineered one enzyme or more than one enzyme at a time. In addition, a nucleic acid molecule encoding one or more enzymes or activities may be used to engineer a host strain of the invention. Preferably, a library of nucleic acid molecules encoding potentially useful enzymes (e.g., chimeric enzymes comprising a catalytically active enzyme fragment ligated in-frame to a heterologous subcellular targeting sequence) is created (e.g., by ligation of sub-libraries comprising enzymatic fragments and subcellular targeting sequences), and a strain having one or more enzymes with optimal activities or producing the most “human-like” or otherwise desirable glycoproteins may be selected by transforming target host cells with one or more members of the library.

In particular, the methods described herein enable one to obtain, in vivo, Man₅GlcNAc₂ structures in high yield, at least transiently, for the purpose of further modifying it to yield complex N-glycans. A successful scheme to obtain suitable Man₅GlcNAc₂ structures in appropriate yields in a host cell, such as a lower eukaryotic organism, generally involves two parallel approaches: (1) reducing high mannose structures made by endogenous mannosyltransferase activities, if any, and (2) removing 1,2-α-mannose by mannosidases to yield high levels of suitable Man₅GlcNAc₂ structures which may be further reacted inside the host cell to form complex, human-like glycoforms.

Accordingly, a first step involves the selection or creation of a eukaryotic host cell, e.g., a lower eukaryote, capable of producing a specific precursor structure of Man₅GlcNAc₂ that is able to accept in vivo GlcNAc by the action of a GlcNAc transferase I (“GnTI”). In one embodiment, the method involves making or using a non-human eukaryotic host cell depleted in a 1,6 mannosyltransferase activity with respect to the N-glycan on a glycoprotein. Preferably, the host cell is depleted in an initiating 1,6 mannosyltransferase activity such as an alpha 1,6 mannosyltransferase activity (see below). Such a host cell will lack one or more enzymes involved in the production of high mannose structures which are undesirable for producing human-like glycoproteins.

One or more enzyme activities are then introduced into such a host cell to produce N-glycans within the host cell characterized by having at least 30 mol % of Man₅GlcNAc₂ (“Man₅”) carbohydrate structures. Man₅GlcNAc₂ structures are necessary for complex N-glycan formation: Man₅GlcNAc₂ must be formed in vivo in a high yield (e.g., in excess of 30%), at least transiently, as subsequent mammalian- and human-like glycosylation reactions require Man₅GlcNAc₂ or a derivative thereof.

This step also requires the formation of a particular isomeric structure of Man₅GlcNAc₂ within the cell at a high yield. While Man₅GlcNAc₂ structures are necessary for complex N-glycan formation, their presence is by no means sufficient. That is because Man₅GlcNAc₂ may occur in different isomeric forms, which may or may not serve as a substrate for GlcNAc transferase I. As most glycosylation reactions are not complete, a particular glycosylated protein generally contains a range of different carbohydrate structures (i.e. glycoforms) on its surface. Thus, the mere presence of trace amounts (i.e., less than 5%) of a particular structure like Man₅GlcNAc₂ is of little practical relevance for producing mammalian- or human-like glycoproteins. It is the formation of a GlcNAc transferase I-accepting Man₅GlcNAc₂ intermediate (FIG. 1B) in high yield (i.e., above 30%), which is required. The formation of this intermediate is necessary to enable subsequent in vivo synthesis of complex N-glycans on glycosylated proteins of interest (target proteins).

Accordingly, some or all of the Man₅GlcNAc₂ produced by the selected host cell must be a productive substrate for enzyme activities along a mammalian glycosylation pathway, e.g., can serve as a substrate for a GlcNAc transferase I activity in vivo, thereby forming the human-like N-glycan intermediate GlcNAcMan₅GlcNAc₂ in the host cell. In a preferred embodiment, at least 10%, more preferably at least 30% and most preferably 50% or more of the Man₅GlcNAc₂ intermediate produced in the host cell of the invention is a productive substrate for GnTI in vivo. It is understood that if, for example, GlcNAcMan₅GlcNAc₂ is produced at 10% and Man₅GlcNAc₂ is produced at 25% on a target protein, the total amount of transiently produced Man₅GlcNAc₂ is 35% because GlcNAcMan₅GlcNAc₂ is a product of Man₅GlcNAc₂.

As described later herein, N-glycans having a terminal GlcNAc residue, such as GlcNAcMan₅GlcNAc₂ can serve as a substrate for sequential addition of galactose [e.g., UDP-galactosyltransferase] and sialic acid [e.g., sialyltransferase, CMP-Sia] to produce the hybrid N-glycans of the present invention.

One of ordinary skill in the art can select host cells from nature, e.g., existing fungi or other lower eukaryotes that produce significant levels of Man₅GlcNAc₂ in vivo. As yet, however, no lower eukaryote has been shown to provide such structures in vivo in excess of 1.8% of the total N-glycans (see e.g. Maras, 1997). Alternatively, such host cells may be genetically engineered to produce the Man₅GlcNAc₂ structure in vivo. Methods such as those described in U.S. Pat. No. 5,595,900 may be used to identify the absence or presence of particular glycosyltransferases, mannosidases and sugar nucleotide transporters in a target host cell or organism of interest.

Inactivation of Undesirable Host Cell Glycosylation Enzymes

The methods of the invention are directed to making host cells that produce glycoproteins having altered, and preferably human-like, N-glycan structures. In a preferred embodiment, the methods are directed to making host cells in which oligosaccharide precursors are enriched in Man₅GlcNAc₂. Preferably, a eukaryotic host cell is used that does not express one or more enzymes involved in the production of high mannose structures. Such a host cell may be found in nature or may be engineered, e.g., starting with or derived from one of many such mutants already described in yeasts. Thus, depending on the selected host cell, one or a number of genes that encode enzymes known to be characteristic of non-human glycosylation reactions may have to be deleted. Such genes and their corresponding proteins have been extensively characterized in a number of lower eukaryotes (e.g., S. cerevisiae, T. reesei, A. nidulans etc.), thereby providing a list of known glycosyltransferases in lower eukaryotes, their activities and their respective genetic sequence. These genes are likely to be selected from the group of mannosyltransferases e.g. 1,3 mannosyltransferases (e.g. MNN1 in S. cerevisiae) (Graham, 1991), 1,2 mannosyltransferases (e.g. KTR/KRE family from S. cerevisiae), 1,6 mannosyltransferases (OCH1 from S. cerevisiae), mannosylphosphate transferases and their regulators (MNN4 and MNN6 from S. cerevisiae) and additional enzymes that are involved in aberrant, i.e. non human, glycosylation reactions. Many of these genes have in fact been deleted individually giving rise to viable phenotypes with altered glycosylation profiles. Examples are shown in Table 1.

Preferred lower eukaryotic host cells of the invention, as described herein to exemplify the required manipulation steps, are hypermannosylation-minus (och1) mutants of Pichia pastoris or K. lactis. Like other lower eukaryotes, P. pastoris processes Man₉GlcNAc₂ structures in the ER with an α-1,2-mannosidase to yield Man₈GlcNAc₂ (FIG. 1A). Through the action of several mannosyltransferases, this structure is then converted to hypermannosylated structures (Man_(>9)GlcNAc₂), also known as mannans. In addition, it has been found that P. pastoris is able to add non-terminal phosphate groups, through the action of mannosylphosphate transferases, to the carbohydrate structure. This differs from the reactions performed in mammalian cells, which involve the removal rather than addition of mannose sugars. It is of particular importance to eliminate the ability of the eukaryotic host cell, e.g., fungus, to hypermannosylate an existing Man₈GlcNAc₂ structure. This can be achieved by either selecting for a host cell that does not hypermannosylate or by genetically engineering such a cell.

Genes that are involved in the hypermannosylation process have been identified, e.g., in Pichia pastoris, and by creating mutations in these genes, one can reduce the production of “undesirable” glycoforms. Such genes can be identified by homology to existing mannosyltransferases or their regulators (e.g., OCH1, MNN4, MNN6, MNN1) found in other lower eukaryotes such as C. albicans, Pichia angusta or S. cerevisiae or by mutagenizing the host strain and selecting for a glycosylation phenotype with reduced mannosylation. Based on homologies amongst known mannosyltransferases and mannosylphosphate transferases, one may either design PCR primers (SEQ ID NOS: 7, 8, 47 and 4 left to right, respectively, in order of appearance) (examples of which are shown in Table 2), or use genes or gene fragments encoding such enzymes as probes to identify homologs in DNA libraries of the target or a related organism. Alternatively, one may identify a functional homolog having mannosyltransferase activity by its ability to complement particular glycosylation phenotypes in related organisms.

TABLE 2 PCR Primers Target Gene(s) in PCR primer A PCR primer B P. pastoris Homologs ATGGCGAAGGCAG TTAGTCCTTCCAAC 1,6- OCH1 S. cerevisiae, ATGGCAGT (SEQ TTCCTTC (SEQ mannosyltransferase Pichia albicans ID NO: 1) ID NO: 2) TAYTGGMGNGTNG GCRTCNCCCCANCK 1,2 KTR/KRE family, ARCYNGAYATHAA YTCRTA (SEQ ID mannosyltransferases S. cerevisiae (SEQ ID NO: NO: 4) 3) Legend: M = A or C, R = A or G, W = A or T, S = C or G, Y = C or T, K = G or T, V = A or C or G, H = A or C or T, D = A or G or T, B = C or G or T, N = G or A or T or C.

To obtain the gene or genes encoding 1,6-mannosyltransferase activity in P. pastoris, for example, one would carry out the following steps: OCH1 mutants of S. cerevisiae are temperature sensitive and are slow growers at elevated temperatures. One can thus identify functional homologs of OCH1 in P. pastoris by complementing an OCH1 mutant of S. cerevisiae with a P. pastoris DNA or cDNA library. Mutants of S. cerevisiae are available, e.g., from Stanford University and are commercially available from ResGen, an Invitrogen Corp. (Carlsbad, Calif.). Mutants that display a normal growth phenotype at elevated temperature, after having been transformed with a P. pastoris DNA library, are likely to carry an OCH1 homolog of P. pastoris. Such a library can be created by partially digesting chromosomal DNA of P. pastoris with a suitable restriction enzyme and, after inactivating the restriction enzyme, ligating the digested DNA into a suitable vector, which has been digested with a compatible restriction enzyme.

Suitable vectors include, e.g., pRS314, a low copy (CEN6/ARS4) plasmid based on pBluescript containing the Trp1 marker (Sikorski, 1989) and pFL44S, a high copy (2μ) plasmid based on a modified pUC19 containing the URA3 marker (Bonneaud, 1991). Such vectors are commonly used by academic researchers and similar vectors are available from a number of different vendors (e.g., Invitrogen (Carlsbad, Calif.); Pharmacia (Piscataway, N.J.); New England Biolabs (Beverly, Mass.)). Further examples include pYES/GS, 2μ origin of replication based yeast expression plasmid from Invitrogen, or Yep24 cloning vehicle from New England Biolabs.

After ligation of the chromosomal DNA and the vector, one may transform the DNA library into a strain of S. cerevisiae with a specific mutation and select for the correction of the corresponding phenotype. After sub-cloning and sequencing the DNA fragment that is able to restore the wild-type phenotype, one may use this fragment to eliminate the activity of the gene product encoded by OCH1 in P. pastoris using in vivo mutagenesis and/or recombination techniques well-known to those skilled in the art.

Alternatively, if the entire genomic sequence of a particular host cell, e.g., fungus, of interest is known, one may identify such genes simply by searching publicly available DNA databases, which are available from several sources, such as NCBI, Swissprot. For example, by searching a given genomic sequence or database with sequences from a known 1,6 mannosyltransferase gene (e.g., OCH1 from S. cerevisiae), one can identify genes of high homology in such a host cell genome which may (but do not necessarily) encode proteins that have 1,6-mannosyltransferase activity. Nucleic acid sequence homology alone is not enough to prove, however, that one has identified and isolated a homolog encoding an enzyme having the same activity. To date, for example, no data exist to show that an OCH1 deletion in P. pastoris eliminates the crucial initiating 1,6-mannosyltransferase activity. (Martinet, 1998; WO 02/00856 A2). Thus, no data prove that the P. pastoris OCH1 gene homolog actually encodes that function. That demonstration is provided for the first time herein.

Homologs to several S. cerevisiae mannosyltransferases have been identified in P. pastoris using these approaches. Homologous genes often have similar functions to genes involved in the mannosylation of proteins in S. cerevisiae and thus their deletion may be used to manipulate the glycosylation pattern in P. pastoris or, by analogy, in any other host cell, e.g., fungus, plant, insect or animal cells, with similar glycosylation pathways.

The creation of gene knock-outs, once a given target gene sequence has been determined, is a well-established technique in the art and can be carried out by one of ordinary skill in the art (see, e.g., Rothstein, 1991). The choice of a host organism may be influenced by the availability of good transformation and gene disruption techniques.

If several mannosyltransferases are to be knocked out, the method developed by Alani, 1987, for example, enables the repeated use of a selectable marker, e.g., the URA3 marker in yeast, to sequentially eliminate all undesirable endogenous mannosyltransferase activity. This technique has been refined by others but basically involves the use of two repeated DNA sequences, flanking a counter selectable marker. For example: URA3 may be used as a marker to ensure the selection of a transformants that have integrated a construct. By flanking the URA3 marker with direct repeats one may first select for transformants that have integrated the construct and have thus disrupted the target gene. After isolation of the transformants, and their characterization, one may counter select in a second round for those that are resistant to 5-fluoroorotic acid (5-FOA). Colonies that are able to survive on plates containing 5-FOA have lost the URA3 marker again through a crossover event involving the repeats mentioned earlier. This approach thus allows for the repeated use of the same marker and facilitates the disruption of multiple genes without requiring additional markers. Similar techniques for sequential elimination of genes adapted for use in another eukaryotic host cells with other selectable and counter-selectable markers may also be used.

Eliminating specific mannosyltransferases, such as 1,6 mannosyltransferase (OCH1) or mannosylphosphate transferases (MNN6, or genes complementing lbd mutants) or regulators (MNN4) in P. pastoris enables one to create engineered strains of this organism which synthesize primarily Man₈GlcNAc₂ and which can be used to further modify the glycosylation pattern to resemble more complex glycoform structures, e.g., those produced in mammalian, e.g., human cells. A preferred embodiment of this method utilizes DNA sequences encoding biochemical glycosylation activities to eliminate similar or identical biochemical functions in P. pastoris to modify the glycosylation structure of glycoproteins produced in the genetically altered P. pastoris strain.

Methods used to engineer the glycosylation pathway in yeasts as exemplified herein can be used in filamentous fungi to produce a preferred substrate for subsequent modification. Strategies for modifying glycosylation pathways in A. niger and other filamentous fungi, for example, can be developed using protocols analogous to those described herein for engineering strains to produce human-like glycoproteins in yeast. Undesired gene activities involved in 1,2 mannosyltransferase activity, e.g., KTR/KRE homologs, are modified or eliminated. A filamentous fungus, such as Aspergillus, is a preferred host because it lacks the 1,6 mannosyltransferase activity and as such, one would not expect a hypermannosylating gene activity, e.g. OCH1, in this host. By contrast, other desired activities (e.g., α-1,2-mannosidase, UDP-GlcNAc transporter, glycosyltransferase (GnT), galactosyltransferase (GalT) and sialyltransferase (ST)) involved in glycosylation are introduced into the host using the targeting methods of the invention.

Engineering or Selecting Hosts Having Diminished Initiating α-1,6 Mannosyltransferase Activity

In a preferred embodiment, the method of the invention involves making or using a host cell which is diminished or depleted in the activity of an initiating α-1,6-mannosyltransferase, i.e., an initiation specific enzyme that initiates outer chain mannosylation on the α-1,3 arm of the Man₃GlcNAc₂ core structure. In S. cerevisiae, this enzyme is encoded by the OCH1 gene. Disruption of the OCH1 gene in S. cerevisiae results in a phenotype in which N-linked sugars completely lack the poly-mannose outer chain. Previous approaches for obtaining mammalian-type glycosylation in fungal strains have required inactivation of OCH1 (see, e.g., Chiba, 1998). Disruption of the initiating α-1,6-mannosyltransferase activity in a host cell of the invention may be optional, however (depending on the selected host cell), as the Och1p enzyme requires an intact Man₈GlcNAc₂ for efficient mannose outer chain initiation. Thus, host cells selected or produced according to this invention which accumulate oligosaccharides having seven or fewer mannose residues may produce hypoglycosylated N-glycans that will likely be poor substrates for Och1p (see, e.g., Nakayama, 1997).

The OCH1 gene was cloned from P. pastoris (Example 1) and K. lactis (Example 18), as described. The nucleic acid and amino acid sequences of the OCH1 gene from K. lactis are set forth in SEQ ID NOS: 41 and 42. Using gene-specific primers, a construct was made from each clone to delete the OCH1 gene from the genome of P. pastoris and K. lactis (Examples 1 and 18, respectively). Host cells depleted in initiating α-1,6-mannosyltransferase activity and engineered to produce N-glycans having a Man₅GlcNAc₂ carbohydrate structure were thereby obtained (see, e.g., FIGS. 5 and 6; Examples 11 and 18).

Thus, in another embodiment, the invention provides an isolated nucleic acid molecule having a nucleic acid sequence comprising or consisting of at least forty-five, preferably at least 50, more preferably at least 60 and most preferably 75 or more nucleotide residues of the K. lactis OCH1 gene (SEQ ID NO: 41), and homologs, variants and derivatives thereof. The invention also provides nucleic acid molecules that hybridize under stringent conditions to the above-described nucleic acid molecules. Similarly, isolated polypeptides (including muteins, allelic variants, fragments, derivatives, and analogs) encoded by the nucleic acid molecules of the invention are provided. Also provided are vectors, including expression vectors, which comprise the above nucleic acid molecules of the invention, as described further herein. Similarly, host cells transformed with the nucleic acid molecules or vectors of the invention are provided.

Host Cells Enriched in Man₅GlcNAc₂

A preferred host cell of the invention is a lower eukaryotic cell, e.g., yeast, a unicellular and multicellular or filamentous fungus. However, a wide variety of host cells are envisioned as being useful in the methods of the invention. Plant cells or insect cells, for instance, may be engineered to express a human-like glycoprotein according to the invention (Examples 19 and 20). Likewise, a variety of non-human, mammalian host cells may be altered to express more human-like or otherwise altered glycoproteins using the methods of the invention. As one of skill in the art will appreciate, any eukaryotic host cell (including a human cell) may be used in conjunction with a library of the invention to express one or more chimeric proteins which is targeted to a subcellular location, e.g., organelle, in the host cell where the activity of the protein is modified, and preferably is enhanced. Such a protein is preferably—but need not necessarily be—an enzyme involved in protein glycosylation, as exemplified herein. It is envisioned that any protein coding sequence may be targeted and selected for modified activity in a eukaryotic host cell using the methods described herein.

Lower eukaryotes that are able to produce glycoproteins having the attached N-glycan Man₅GlcNAc₂ are particularly useful because (a) lacking a high degree of mannosylation (e.g. greater than 8 mannoses per N-glycan, or especially 30-40 mannoses), they show reduced immunogenicity in humans; and (b) the N-glycan is a substrate for further glycosylation reactions to form an even more human-like glycoform, e.g., by the action of GlcNAc transferase I (FIG. 1B; β1, 2 GnTI) to form GlcNAcMan₅GlcNAc₂. A yield is obtained of greater than 30 mole %, more preferably a yield of 50-100 mole %, glycoproteins with N-glycans having a Man₅GlcNAc₂ structure. In a preferred embodiment, more than 50% of the Man₅GlcNAc₂ structure is shown to be a substrate for a GnTI activity and can serve as such a substrate in vivo. Lower eukaryotes may also be useful host cells of the invention because they typically lack (unless otherwise engineered to add) galactose, fucose and N-acetylglucosamine. Thus, recombinant proteins lacking, e.g., fucose, may be made in these host cells.

Preferred lower eukaryotes of the invention include but are not limited to: any Pichia sp., including but limited to: Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis and Pichia methanolica; any Saccharomyces sp including but not limited to: Saccharomyces cerevisiae, Hansenula polymorpha; any Kluyveromyces sp. including but not limited to: Kluyveromyces lactis; Candida albicans; Aspergillus nidulans; Aspergillus niger; Aspergillus oryzae; Trichoderma reseei; Chrysosporium lucknowense; any Fusarium sp. including but not limited to: Fusarium gramineum and Fusarium venenatum; and Neurospora crassa.

In each above embodiment, the method is directed to making a host cell in which the oligosaccharide precursors are enriched in Man₅GlcNAc₂. These structures are desirable because they may then be processed by treatment in vitro, for example, using the method of U.S. Pat. No. 5,834,251. In a preferred embodiment, however, precursors enriched in Man₅GlcNAc₂ are processed by at least one further glycosylation reaction in vivo—with glycosidases (e.g., α-mannosidases) and glycosyltransferases (e.g., GnTI)—to produce human-like N-glycans. Oligosaccharide precursors enriched in Man₅GlcNAc₂, for example, are preferably processed to those having GlcNAcMan_(X)GlcNAc₂ core structures, wherein X is 3, 4 or 5, and is preferably 3. N-glycans having a GlcNAcMan_(X)GlcNAc₂ core structure where X is greater than 3 may be converted to GlcNAcMan₃GlcNAc₂, e.g., by treatment with an α-1,3 and/or α-1,6 mannosidase activity, where applicable. Additional processing of GlcNAcMan₃GlcNAc₂ by treatment with glycosyltransferases (e.g., GnTII) produces GlcNAc₂Man₃GlcNAc₂ core structures which may then be modified, as desired, e.g., by ex vivo treatment or by heterologous expression in the host cell of additional glycosylation enzymes, including glycosyltransferases, sugar transporters and mannosidases (see below), to comprise specific human-like N-glycans, as desired.

Preferred human-like glycoproteins which may be produced according to the invention include those that comprise N-glycans having seven or fewer, or three or fewer, mannose residues; and that comprise one or more sugars selected from the group consisting of galactose, GlcNAc, sialic acid, and fucose. Other preferred human-like glycoproteins which may be produced according to the invention lack fucose.

Formation of Complex N-Glycans

Formation of complex N-glycan synthesis is a sequential process by which specific sugar residues are removed and attached to the core oligosaccharide structure. In higher eukaryotes, this is achieved by having the substrate sequentially exposed to various processing enzymes. These enzymes carry out specific reactions depending on their particular location within the entire processing cascade. This “assembly line” consists of ER, early, medial and late Golgi, and the trans Golgi network all with their specific processing environment. To re-create the processing of human glycoproteins in the Golgi and ER of lower eukaryotes, numerous enzymes (e.g. glycosyltransferases, glycosidases, phosphatases and transporters) have to be expressed and specifically targeted to these organelles, and preferably, in a location so that they function most efficiently in relation to their environment as well as to other enzymes in the pathway.

Because one goal of the methods described herein is to achieve a robust protein production strain that is able to perform well in an industrial fermentation process, the integration of multiple genes into the host cell chromosome involves careful planning. As described above, one or more genes which encode enzymes known to be characteristic of non-human glycosylation reactions are preferably deleted. The engineered cell strain is transformed with a range of different genes encoding desired activities, and these genes are transformed in a stable fashion, thereby ensuring that the desired activity is maintained throughout the fermentation process.

Any combination of the following enzyme activities may be engineered singly or multiply into the host using methods of the invention: sialyltransferases, mannosidases, fucosyltransferases, galactosyltransferases, GlcNAc transferases, ER and Golgi specific transporters (e.g. syn- and antiport transporters for UDP-galactose and other precursors), other enzymes involved in the processing of oligosaccharides, and enzymes involved in the synthesis of activated oligosaccharide precursors such as UDP-galactose and CMP-N-acetylneuraminic acid. Preferably, enzyme activities are introduced on one or more nucleic acid molecules (see also below). Nucleic acid molecules may be introduced singly or multiply, e.g., in the context of a nucleic acid library such as a combinatorial library of the invention. It is to be understood, however, that single or multiple enzymatic activities may be introduced into a host cell in any fashion, including but not limited to protein delivery methods and/or by use of one or more nucleic acid molecules without necessarily using a nucleic acid library or combinatorial library of the invention.

Expression of Glycosyltransferases to Produce Complex N-Glycans

With DNA sequence information, the skilled artisan can clone DNA molecules encoding one or more GnT activities such as GnTI, II, III, IV or IV (e.g., Examples 3 and 4); galactosyltransferase activities (Example 4); and/or sialyltransferase activities (Examples 6 and 17). Using standard techniques well-known to those of skill in the art, nucleic acid molecules encoding one or more of the above described enzymes (or encoding catalytically active fragments thereof) may be inserted into appropriate expression vectors under the transcriptional control of promoters and other expression control sequences capable of driving transcription in a selected host cell of the invention, e.g., a fungal host such as Pichia sp., Kluyveromyces sp. and Aspergillus sp., as described herein, such that one or more of these mammalian enzymes may be actively expressed in a host cell of choice for production of a human-like complex glycoprotein (e.g., Examples 15, 16, 17, 19 and 20).

Several individual glycosyltransferases have been cloned and expressed in S. cerevisiae (GalT, GnTI), Aspergillus nidulans (GnTI) and other fungi, without however demonstrating the desired outcome of “humanization” on the glycosylation pattern of the organisms (Yoshida, 1995; Schwientek, 1995; Kalsner, 1995). It was speculated that the carbohydrate structure required to accept sugars by the action of such glycosyltransferases was not present in sufficient amounts, which most likely contributed to the lack of complex N-glycan formation.

A preferred method of the invention provides the functional expression of a GnT, such as GnTI, in the early or medial Golgi apparatus as well as ensuring a sufficient supply of UDP-GlcNAc (e.g., by expression of a UDP-GlcNAc transporter; see below).

Another preferred method of the invention provides functional expression of a sialyltransferase, such as ST6Gal or ST3Gal, in the Golgi as well as ensuring a sufficient supply of CMP-Sialic acid (e.g., by expression of a CMP-Sialic acid biosynthetic pathway in the host as illustrated in Example 16). Sialyltransferases from many species are known and may be useful in the present invention. Sialyltransferases are described for example, in Harduin-Lepers, 2001; Hardeuin-Lepers, 2005; and Tsji, 1996. The disclosures of all of these references are hereby incorporated herein by reference.

Methods for Providing Sugar Nucleotide Precursors to the Golgi Apparatus

For a glycosyltransferase to function satisfactorily in the Golgi, the enzyme requires a sufficient concentration of an appropriate nucleotide sugar, which is the high-energy donor of the sugar moiety added to a nascent glycoprotein. In humans and other non-human eukaryotic cells, the full range of nucleotide sugar precursors (e.g. UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine, CMP-N-acetylneuraminic acid, UDP-galactose, etc.) are generally synthesized in the cytosol and transported into the Golgi, where they are attached to the core oligosaccharide by glycosyltransferases.

To replicate this process in host cells that do not comprise these nucleotide precursors in the Golgi (e.g., in non-human host cells such as lower eukaryotes), sugar nucleoside specific transporters have to be expressed in the Golgi to ensure adequate levels of nucleoside sugar precursors (Sommers, 1981; Sommers, 1982; Perez, 1987). Nucleotide sugars may be provided to the appropriate compartments, e.g., by expressing in the host cell an exogenous gene encoding a sugar nucleotide transporter. The choice of transporter enzyme is influenced by the nature of the exogenous glycosyltransferase being used. For example, a GlcNAc transferase may require a UDP-GlcNAc transporter, a fucosyltransferase may require a GDP-fucose transporter, a galactosyltransferase may require a UDP-galactose transporter, and a sialyltransferase may require a CMP-sialic acid transporter.

The added transporter protein conveys a nucleotide sugar from the cytosol into the Golgi apparatus, where the nucleotide sugar may be reacted by the glycosyltransferase, e.g. to elongate an N-glycan. The reaction liberates a nucleoside diphosphate or monophosphate, e.g. UDP, GDP, or CMP. Nucleoside monophosphates can be directly exported from the Golgi in exchange for nucleoside triphosphate sugars by an antiport mechanism. Accumulation of a nucleoside diphosphate, however, inhibits the further activity of a glycosyltransferase. As this reaction appears to be important for efficient glycosylation, it is frequently desirable to provide an expressed copy of a gene encoding a nucleotide diphosphatase. The diphosphatase (specific for UDP or GDP as appropriate) hydrolyzes the diphosphonucleoside to yield a nucleoside monophosphate and inorganic phosphate.

Suitable transporter enzymes, which are typically of mammalian origin, are described below. Such enzymes may be engineered into a selected host cell using the methods of the invention (see also Examples 7-10).

In another example, α 2,3- or α 2,6-sialyltransferase caps galactose residues with sialic acid in the trans-Golgi and TGN of humans leading to a mature form of the glycoprotein (FIG. 1B). To reengineer this processing step into a lower eukaryotic host cell and other host cells which naturally lack sialyltransferase activity will require (1) α 2,3- or α 2,6-sialyltransferase activity and (2) a sufficient supply of CMP-N-acetyl neuraminic acid, in the late Golgi (Examples 6, 16 and 17). To obtain sufficient α 2,3-sialyltransferase activity in the secretory pathway (e.g. late Golgi), for example, the catalytic domain of a known sialyltransferase (e.g. from humans) has to be directed to the secretory pathway in lower eukaryotic host cells (see above). Likewise, transporters have to be engineered to allow the transport of CMP-N-acetyl neuraminic acid into the same location of the secretory pathway (e.g. late Golgi). There is currently no indication that host cells such as lower eukaryotic host cells synthesize or can even transport sufficient amounts of CMP-N-acetyl neuraminic acid into the Golgi. Consequently, to ensure the adequate supply of substrate for the corresponding glycosyltransferases, one has to metabolically engineer the production of CMP-sialic acid into these host cells.

UDP-N-Acetylglucosamine Transporter

The cDNA of human UDP-N-acetylglucosamine transporter, which was recognized through a homology search in the expressed sequence tags database (dbEST), has been cloned (Ishida, 1999). The mammalian Golgi membrane transporter for UDP-N-acetylglucosamine was cloned by phenotypic correction with cDNA from canine kidney cells (MDCK) of a recently characterized Kluyveromyces lactis mutant deficient in Golgi transport of the above nucleotide sugar (Guillen, 1998). Results demonstrate that the mammalian Golgi UDP-GlcNAc transporter gene has all of the necessary information for the protein to be expressed and targeted functionally to the Golgi apparatus of yeast and that two proteins with very different amino acid sequences may transport the same solute within the same Golgi membrane (Guillen, 1998).

Accordingly, one may incorporate the expression of a UDP-GlcNAc transporter in a host cell by means of a nucleic acid construct which may contain, for example: (1) a region by which the transformed construct is maintained in the cell (e.g. origin of replication or a region that mediates chromosomal integration), (2) a marker gene that allows for the selection of cells that have been transformed, including counterselectable and recyclable markers such as ura3 or T-urf13 (Soderholm, 2001) or other well characterized selection-markers (e.g., his4, bla, Sh ble etc.), (3) a gene or fragment thereof encoding a functional UDP-GlcNAc transporter, e.g. from K. lactis (Abeijon, 1996), or from H. sapiens (Ishida, 1996), and (4) a promoter activating the expression of the above mentioned localization/catalytic domain fusion construct library.

GDP-Fucose Transporter

The rat liver Golgi membrane GDP-fucose transporter has been identified and purified by Puglielli, L. and C. B. Hirschberg (Puglielli, 1999). The corresponding gene has not been identified; however, N-terminal sequencing can be used for the design of oligonucleotide probes specific for the corresponding gene. These oligonucleotides can be used as probes to clone the gene encoding for GDP-fucose transporter.

UDP-Galactose Transporter

Two heterologous genes, gmal2(+) encoding alpha 1,2-galactosyltransferase (alpha 1,2 GalT) from Schizosaccharomyces pombe and (hUGT2) encoding human UDP-galactose (UDP-Gal) transporter, have been functionally expressed in S. cerevisiae to examine the intracellular conditions required for galactosylation. Correlation between protein galactosylation and UDP-galactose transport activity indicated that an exogenous supply of UDP-Gal transporter, rather than alpha 1,2 GalT played a key role for efficient galactosylation in S. cerevisiae (Kainuma, 1999). Likewise, an UDP-galactose transporter from S. pombe was cloned (Aoki, 1999; Segawa, 1999).

CMP-N-Acetylneuraminic Acid (CMP-Sialic Acid) Transporter

Human CMP-sialic acid transporter (hCST) has been cloned and expressed in Lec 8 CHO cells (Aoki, 1999; Eckhardt, 1997). The functional expression of the murine CMP-sialic acid transporter was achieved in Saccharomyces cerevisiae (Berninsone, 1997). Sialic acid has been found in some fungi, however it is not clear whether the chosen host system will be able to supply sufficient levels of CMP-Sialic acid. Sialic acid can be either supplied in the medium or alternatively fungal pathways involved in sialic acid synthesis can also be integrated into the host genome as described below.

Expression of Diphosphatases

When sugars are transferred onto a glycoprotein, either a nucleoside diphosphate or monophosphate is released from the sugar nucleotide precursors. While monophosphates can be directly exported in exchange for nucleoside triphosphate sugars by an antiport mechanism, diphosphonucleosides (e.g. GDP) have to be cleaved by phosphatases (e.g. GDPase) to yield nucleoside monophosphates and inorganic phosphate prior to being exported. This reaction appears to be important for efficient glycosylation, as GDPase from S. cerevisiae has been found to be necessary for mannosylation. However, the enzyme only has 10% of the activity towards UDP (Berninsone, 1994). Lower eukaryotes often do not have UDP-specific diphosphatase activity in the Golgi as they do not utilize UDP-sugar precursors for glycoprotein synthesis in the Golgi. Schizosaccharomyces pombe, a yeast which adds galactose residues to cell wall polysaccharides (from UDP-galactose), was found to have specific UDPase activity, further suggesting the requirement for such an enzyme (Berninsone, 1994). UDP is known to be a potent inhibitor of glycosyltransferases and the removal of this glycosylation side product is important to prevent glycosyltransferase inhibition in the lumen of the Golgi (Khatara, 1974).

Formation of Hybrid Glycoproteins

With minor modifications, the same “assembly line” of enzymes and glycosylation pathway may be utilized to accomplish the formation of complex N-glycans. The primary difference is that after action of GNT I on the Man5GlcNAc2 core to produce GlcNAcMan5GlcNAc2, it is not necessary that the GlcNAcMan5GlcNAc2 be exposed to the action mannosidase II to form GlcNAcMan3GlcNAc2. See, e.g., Gerngross, WO02/00879 and U.S. Pat. No. 7,029,872.

Methods for Altering N-Glycans in a Host by Expressing a Targeted Enzymatic Activity from a Nucleic Acid Molecule

In one preferred embodiment, the invention provides a method for producing a glycoprotein comprising Man₅GlcNAc₂. In one preferred embodiment, a nucleic acid molecule encoding one or more mannosidase activities involved in the production of Man₅GlcNAc₂ from Man₈GlcNAc₂ or Man₉GlcNAc₂ is introduced into the host.

In another embodiment, the invention provides a method for producing a human-like glycoprotein in a non-human eukaryotic host cell comprising the step of introducing into the host cell one or more nucleic acid molecules that encode an enzyme or enzymes for production of NANA₍₁₋₄₎Gal₍₁₋₄₎GlcNAc₍₁₋₄₎Man₃GlcNAc₂ carbohydrate structure.

The invention additionally relates to methods for making altered glycoproteins in a host cell comprising the step of introducing into the host cell a nucleic acid molecule which encodes one or more glycosylation enzymes or activities. Preferred enzyme activities are selected from the group consisting of UDP-GlcNAc transferase, UDP-galactosyltransferase, GDP-fucosyltransferase, CMP-sialyltransferase, UDP-GlcNAc transporter, UDP-galactose transporter, GDP-fucose transporter, CMP-sialic acid transporter, and nucleotide diphosphatases. In a particularly preferred embodiment, the host is selected or engineered to express two or more enzymatic activities in which the product of one activity increases substrate levels of another activity, e.g., a glycosyltransferase and a corresponding sugar transporter, e.g., GnTI and UDP-GlcNAc transporter activities. In another preferred embodiment, the host is selected or engineered to expresses an activity to remove products which may inhibit subsequent glycosylation reactions, e.g. a UDP- or GDP-specific diphosphatase activity.

Preferred methods of the invention involve expressing one or more enzymatic activities from a nucleic acid molecule in a host cell and comprise the step of targeting at least one enzymatic activity to a desired subcellular location (e.g., an organelle) by forming a fusion protein comprising a catalytic domain of the enzyme and a cellular targeting signal peptide, e.g., a heterologous signal peptide which is not normally ligated to or associated with the catalytic domain. The fusion protein is encoded by at least one genetic construct (“fusion construct”) comprising a nucleic acid fragment encoding a cellular targeting signal peptide ligated in the same translational reading frame (“in-frame”) to a nucleic acid fragment encoding an enzyme (e.g., glycosylation enzyme), or catalytically active fragment thereof.

The targeting signal peptide component of the fusion construct or protein is preferably derived from a member of the group consisting of: membrane-bound proteins of the ER or Golgi, retrieval signals, Type II membrane proteins, Type I membrane proteins, membrane spanning nucleotide sugar transporters, mannosidases, sialyltransferases, glucosidases, mannosyltransferases and phosphomannosyltransferases.

The catalytic domain component of the fusion construct or protein is preferably derived from a glycosidase, mannosidase or a glycosyltransferase activity derived from a member of the group consisting of GnTI, GnTII, GnTIII, GnTIV, GnTV, GnTVI, GalT, Fucosyltransferase and Sialyltransferase. The catalytic domain preferably has a pH optimum within 1.4 pH units of the average pH optimum of other representative enzymes in the organelle in which the enzyme is localized, or has optimal activity at a pH between 5.1 and 8.0.

Selecting a Glycosylation Enzyme: pH Optima and Subcellular Localization

In one embodiment of the invention, a human-like glycoprotein is made efficiently in a non-human eukaryotic host cell by introducing into a subcellular compartment of the cell a glycosylation enzyme selected to have a pH optimum similar to the pH optima of other enzymes in the targeted subcellular compartment. For example, most enzymes that are active in the ER and Golgi apparatus of S. cerevisiae have pH optima that are between about 6.5 and 7.5 (see Table 3). Because the glycosylation of proteins is a highly evolved and efficient process, the internal pH of the ER and the Golgi is likely also in the range of about 6-8. All previous approaches to reduce mannosylation by the action of recombinant mannosidases in fungal hosts, however, have introduced enzymes that have a pH optimum of around pH 5.0 (Martinet, 1998 and Chiba, 1998). At pH 7.0, the in vitro determined activity of those mannosidases is reduced to less than 10%, which is likely insufficient activity at their point of use, namely, the ER and early Golgi, for the efficient in vivo production of Man₅GlcNAc₂ on N-glycans.

Accordingly, a preferred embodiment of this invention targets a selected glycosylation enzyme (or catalytic domain thereof), e.g., an α-mannosidase, to a subcellular location in the host cell (e.g., an organelle) where the pH optimum of the enzyme or domain is within 1.4 pH units of the average pH optimum of other representative marker enzymes localized in the same organelle(s). The pH optimum of the enzyme to be targeted to a specific organelle should be matched with the pH optimum of other enzymes found in the same organelle to maximize the activity per unit enzyme obtained. Table 3 summarizes the activity of mannosidases from various sources and their respective pH optima. Table 4 summarizes their typical subcellular locations.

TABLE 3 Mannosidases And Their pH Optimum. pH Source Enzyme optimum Reference Aspergillus saitoi α-1,2-mannosidase 5.0 Ichishima, 1999 Trichoderma α-1,2-mannosidase 5.0 Maras, 2000 reesei Penicillium α-D-1,2- 5.0 Yoshida, 1993 citrinum mannosidase C. elegans α-1,2-mannosidase 5.5 see FIG. 11 Aspergillus α-1,2-mannosidase 6.0 Eades and Hintz, nidulans 2000 Homo sapiens α-1,2-mannosidase 6.0 IA(Golgi) Homo sapiens IB α-1,2-mannosidase 6.0 (Golgi) Lepidopteran Type I α-1,2-Man₆- 6.0 Ren, 1995 insect cells mannosidase Homo sapiens α-D-mannosidase 6.0 Chandrasekaran, 1984 Xanthomonas α-1,2,3-mannosidase 6.0 U.S. Pat. No. manihotis 6,300,113 Mouse IB (Golgi) α-1,2-mannosidase 6.5 Schneikert, 1994 Bacillus sp. α-D-1,2- 7.0 Maruyama, 1994 (secreted) mannosidase

In a preferred embodiment, a particular enzyme or catalytic domain is targeted to a subcellular location in the host cell by means of a chimeric fusion construct encoding a protein comprising a cellular targeting signal peptide not normally associated with the enzymatic domain. Preferably, an enzyme or domain is targeted to the ER, the early, medial or late Golgi of the trans Golgi apparatus of the host cell.

In a more preferred embodiment, the targeted glycosylation enzyme is a glycosyltransferase or a glycosidase (such as a mannosidase). In an especially preferred embodiment, an enzyme having sialyltransferase activity is targeted to the late Golgi, where the early reactions of glycosylation occur. While the methods disclosed herein are useful for producing a human-like glycoprotein in a non-human host cell, it will be appreciated that the methods discussed herein are also useful more generally for modifying carbohydrate profiles of a glycoprotein in any eukaryotic host cell, including human host cells. See Gerngross, WO02/00879.

Targeting sequences which mediate retention of proteins in certain organelles of the host cell secretory pathway are well-known and described in the scientific literature and public databases, as discussed in more detail below with respect to libraries for selection of targeting sequences and targeted enzymes. Such subcellular targeting sequences may be used alone or in combination to target a selected glycosylation enzyme (or catalytic domain thereof) to a particular subcellular location in a host cell, i.e., especially to one where the enzyme will have enhanced or optimal activity based on pH optima or the presence of other stimulatory factors.

When one attempts to trim high mannose structures to yield Man₅GlcNAc₂ in the ER or the Golgi apparatus of a host cell such as S. cerevisiae, for example, one may choose any enzyme or combination of enzymes that (1) has a sufficiently close pH optimum (i.e. between pH 5.2 and pH 7.8), and (2) is known to generate, alone or in concert, the specific isomeric Man₅GlcNAc₂ structure required to accept subsequent addition of GlcNAc by GnTI. Any enzyme or combination of enzymes that is shown to generate a structure that can be converted to GlcNAcMan₅GlcNAc₂ by GnTI in vitro would constitute an appropriate choice. This knowledge may be obtained from the scientific literature or experimentally.

For example, one may determine whether a potential mannosidase can convert Man₈GlcNAc₂-2AB (2-aminobenzamide) to Man₅GlcNAc₂-AB and then verify that the obtained Man₅GlcNAc₂-2AB structure can serve a substrate for GnTI and UDP-GlcNAc to give GlcNAcMan₅GlcNAc₂ in vitro. Mannosidase IA from a human or murine source, for example, would be an appropriate choice (see, e.g., Example 11). Examples described herein utilize 2-aminobenzamide labeled N-linked oligomannose followed by HPLC analysis to make this determination.

TABLE 4 Cellular Location And pH Optima Of Various Glycosylation-Related Enzymes Of S. cerevisiae. Gene Activity Location pH optimum Reference(s) KTR1 α- 1,2 Golgi 7.0 Romero, 1997 mannosyltransferase MNS1 α- 1,2- mannosidase ER 6.5 CWH41 glucosidase I ER 6.8 — mannosyltransferase Golgi 7-8 Lehele, 1974 KRE2 α- 1,2 Golgi 6.5-9.0 Romero, 1997 mannosyltransferase

Accordingly, a glycosylation enzyme such as an α-1,2-mannosidase enzyme used according to the invention has an optimal activity at a pH of between 5.1 and 8.0. In a preferred embodiment, the enzyme has an optimal activity at a pH of between 5.5 and 7.5. The C. elegans mannosidase enzyme, for example, works well in the methods of the invention and has an apparent pH optimum of about 5.5).

The experiment which illustrates the pH optimum for an α-1,2-mannosidase enzyme is described in Example 14. A chimeric fusion protein BB27-2 (Saccharomyces MNN10 (s)/C. elegans mannosidase IB Δ31), which leaks into the medium was subjected to various pH ranges to determine the optimal activity of the enzyme. The results of the experiment show that the α-1,2-mannosidase has an optimal pH of about 5.5 for its function (FIG. 11).

In a preferred embodiment, a single cloned mannosidase gene is expressed in the host organism. However, in some cases it may be desirable to express several different mannosidase genes, or several copies of one particular gene, in order to achieve adequate production of Man₅GlcNAc₂. In cases where multiple genes are used, the encoded mannosidases preferably all have pH optima within the preferred range of about 5.1 to about 8.0, or especially between about 5.5 and about 7.5. Preferred mannosidase activities include α-1,2-mannosidases derived from mouse, human, Lepidoptera , Aspergillus nidulans, or Bacillus sp., C. elegans, D. melanogaster, P. citrinum, X. laevis or A. nidulans.

In Vivo Alteration of Host Cell Glycosylation Using a Combinatorial DNA Library

Certain methods of the invention are preferably (but need not necessarily be) carried out using one or more nucleic acid libraries. An exemplary feature of a combinatorial nucleic acid library of the invention is that it comprises sequences encoding cellular targeting signal peptides and sequences encoding proteins to be targeted (e.g., enzymes or catalytic domains thereof, including but not limited to those which mediate glycosylation).

In one embodiment, a combinatorial nucleic acid library comprises: (a) at least two nucleic acid sequences encoding different cellular targeting signal peptides; and (b) at least one nucleic acid sequence encoding a polypeptide to be targeted. In another embodiment, a combinatorial nucleic acid library comprises: (a) at least one nucleic acid sequence encoding a cellular targeting signal peptide; and (b) at least two nucleic acid sequences encoding a polypeptide to be targeted into a host cell. As described further below, a nucleic acid sequence derived from (a) and a nucleic acid sequence derived from (b) are ligated to produce one or more fusion constructs encoding a cellular targeting signal peptide functionally linked to a polypeptide domain of interest. One example of a functional linkage is when the cellular targeting signal peptide is ligated to the polypeptide domain of interest in the same translational reading frame (“in-frame”).

In a preferred embodiment, a combinatorial DNA library expresses one or more fusion proteins comprising cellular targeting signal peptides ligated in-frame to catalytic enzyme domains. The encoded fusion protein preferably comprises a catalytic domain of an enzyme involved in mammalian- or human-like modification of N-glycans. In a more preferred embodiment, the catalytic domain is derived from an enzyme selected from the group consisting of mannosidases, glycosyltransferases and other glycosidases which is ligated in-frame to one or more targeting signal peptides. The enzyme domain may be exogenous and/or endogenous to the host cell. A particularly preferred signal peptide is one normally associated with a protein that undergoes ER to Golgi transport.

The combinatorial DNA library of the present invention may be used for producing and localizing in vivo enzymes involved in mammalian- or human-like N-glycan modification. The fusion constructs of the combinatorial DNA library are engineered so that the encoded enzymes are localized in the ER, Golgi or the trans-Golgi network of the host cell where they are involved in producing particular N-glycans on a glycoprotein of interest. Localization of N-glycan modifying enzymes of the present invention is achieved through an anchoring mechanism or through protein-protein interaction where the localization peptide constructed from the combinatorial DNA library localizes to a desired organelle of the secretory pathway such as the ER, Golgi or the trans Golgi network.

An example of a useful N-glycan, which is produced efficiently and in sufficient quantities for further modification by human-like (complex) glycosylation reactions is Man₅GlcNAc₂. A sufficient amount of Man₅GlcNAc₂ is needed on a glycoprotein of interest for further human-like processing in vivo (e.g., more than 30 mole %). The Man₅GlcNAc₂ intermediate may be used as a substrate for further N-glycan modification to produce GlcNAcMan₅GlcNAc₂ (FIG. 1B; see above). Accordingly, the combinatorial DNA library of the present invention may be used to produce enzymes which subsequently produce GlcNAcMan₅GlcNAc₂, or other desired complex N-glycans, in a useful quantity.

A further aspect of the fusion constructs produced using the combinatorial DNA library of the present invention is that they enable sufficient and often near complete intracellular N-glycan trimming activity in the engineered host cell. Preferred fusion constructs produced by the combinatorial DNA library of the invention encode a glycosylation enzyme, e.g., a mannosidase, which is effectively localized to an intracellular host cell compartment and thereby exhibits very little and preferably no extracellular activity. The preferred fusion constructs of the present invention that encode a mannosidase enzyme are shown to localize where the N-glycans are modified, namely, the ER and the Golgi. The fusion enzymes of the present invention are targeted to such particular organelles in the secretory pathway where they localize and act upon N-glycans such as Man₈GlcNAc₂ to produce Man₅GlcNAc₂ on a glycoprotein of interest.

Enzymes produced by the combinatorial DNA library of the present invention can modify N-glycans on a glycoprotein of interest as shown for K3 or IFN-β proteins expressed in P. pastoris, as shown in FIG. 5 and FIG. 6, respectively (see also Examples 2 and 11). It is, however, appreciated that other types of glycoproteins, without limitation, including erythropoietin, cytokines such as interferon-α, interferon-β, interferon-γ, interferon-ω, and granulocyte-CSF, coagulation factors such as factor VIII, factor IX, and human protein C, soluble IgE receptor α-chain, IgG, IgG fragments, IgM, interleukins, urokinase, chymase, and urea trypsin inhibitor, IGF-binding protein, epidermal growth factor, growth hormone-releasing factor, annexin V fusion protein, angiostatin, vascular endothelial growth factor-2, myeloid progenitor inhibitory factor-1, osteoprotegerin, α-1 antitrypsin, DNase II, α-feto proteins, AAT, rhTBP-1 (onercept, aka TNF Binding protein 1), TACI-Ig (transmembrane activator and calcium modulator and cyclophilin ligand interactor), FSH (follicle stimulating hormone), GM-CSF, GLP-1 w/ and w/o FC (glucagon like protein 1) IL-1 receptor agonist, sTNFr (enbrel, aka soluble TNF receptor Fc fusion) ATIII, rhThrombin, glucocerebrosidase and CTLA4-Ig (Cytotoxic T Lymphocyte associated Antigen 4-Ig) may be glycosylated in this way.

Constructing a Combinatorial DNA Library of Fusion Constructs

A combinatorial DNA library of fusion constructs features one or more cellular targeting signal peptides (“targeting peptides”) generally derived from N-terminal domains of native proteins (e.g., by making C-terminal deletions). Some targeting peptides, however, are derived from the C-terminus of native proteins (e.g. SEC12). Membrane-bound proteins of the ER or the Golgi are preferably used as a source for targeting peptide sequences. These proteins have sequences encoding a cytosolic tail (ct), a transmembrane domain (tmd) and a stem region (sr) which are varied in length. These regions are recognizable by protein sequence alignments and comparisons with known homologs and/or other localized proteins (e.g., comparing hydrophobicity plots).

The targeting peptides are indicated herein as short (s), medium (m) and long (l) relative to the parts of a type II membrane. The targeting peptide sequence indicated as short (s) corresponds to the transmembrane domain (tmd) of the membrane-bound protein. The targeting peptide sequence indicated as long (l) corresponds to the length of the transmembrane domain (tmd) and the stem region (sr). The targeting peptide sequence indicated as medium (m) corresponds to the transmembrane domain (tmd) and approximately half the length of the stem region (sr). The catalytic domain regions are indicated herein by the number of nucleotide deletion with respect to its wild-type glycosylation enzyme.

Sub-Libraries

In some cases a combinatorial nucleic acid library of the invention may be assembled directly from existing or wild-type genes. In a preferred embodiment, the DNA library is assembled from the fusion of two or more sub-libraries. By the in-frame ligation of the sub-libraries, it is possible to create a large number of novel genetic constructs encoding useful targeted protein domains such as those which have glycosylation activities.

Catalytic Domain Sub-Libraries Encoding Glycosylation Activities

One useful sub-library includes DNA sequences encoding enzymes such as glycosidases (e.g., mannosidases), glycosyltransferases (e.g., fucosyl-transferases, galactosyltransferases, glucosyltransferases), GlcNAc transferases and sialyltransferases. Catalytic domains may be selected from the host to be engineered, as well as from other related or unrelated organisms. Mammalian, plant, insect, reptile, algal or fungal enzymes are all useful and should be chosen to represent a broad spectrum of biochemical properties with respect to temperature and pH optima. In a preferred embodiment, genes are truncated to give fragments some of which encode the catalytic domains of the enzymes. By removing endogenous targeting sequences, the enzymes may then be redirected and expressed in other cellular loci.

The choice of such catalytic domains may be guided by the knowledge of the particular environment in which the catalytic domain is subsequently to be active. For example, if a particular glycosylation enzyme is to be active in the late Golgi, and all known enzymes of the host organism in the late Golgi have a certain pH optimum, or the late Golgi is known to have a particular pH, then a catalytic domain is chosen which exhibits adequate, and preferably maximum, activity at that pH, as discussed above.

Targeting Peptide Sequence Sub-Libraries

Another useful sub-library includes nucleic acid sequences encoding targeting signal peptides that result in localization of a protein to a particular location within the ER, Golgi, or trans Golgi network. These targeting peptides may be selected from the host organism to be engineered as well as from other related or unrelated organisms. Generally such sequences fall into three categories: (1) N-terminal sequences encoding a cytosolic tail (ct), a transmembrane domain (tmd) and part or all of a stem region (sr), which together or individually anchor proteins to the inner (lumenal) membrane of the Golgi; (2) retrieval signals which are generally found at the C-terminus such as the HDEL (SEQ ID NO: 5) or KDEL (SEQ ID NO: 6) tetrapeptide; and (3) membrane spanning regions from various proteins, e.g., nucleotide sugar transporters, which are known to localize in the Golgi.

In the first case, where the targeting peptide consists of various elements (ct, tmd and sr), the library is designed such that the ct, the tmd and various parts of the stem region are represented. Accordingly, a preferred embodiment of the sub-library of targeting peptide sequences includes ct, tmd, and/or sr sequences from membrane-bound proteins of the ER or Golgi. In some cases it may be desirable to provide the sub-library with varying lengths of sr sequence. This may be accomplished by PCR using primers that bind to the 5′ end of the DNA encoding the cytosolic region and employing a series of opposing primers that bind to various parts of the stem region.

Still other useful sources of targeting peptide sequences include retrieval signal peptides, e.g. the tetrapeptides HDEL (SEQ ID NO: 5) or KDEL (SEQ ID NO: 6), which are typically found at the C-terminus of proteins that are transported retrograde into the ER or Golgi. Still other sources of targeting peptide sequences include (a) type II membrane proteins, (b) the enzymes listed in Table 3, (c) membrane spanning nucleotide sugar transporters that are localized in the Golgi, and (d) sequences referenced in Table 5. (The HDEL signal in column 1, cell 8 is shown in SEQ ID NO: 5).

TABLE 5 Sources Of Useful Compartmental Targeting Sequences Gene or Location of Gene Sequence Organism Function Product MNSI A. nidulans a-1,2-mannosidase ER MNSI A. niger a-1,2-mannosidase ER MNSI S. cerevisiae a-1,2-mannosidase ER GLSI S. cerevisiae glucosidase ER GLSI A. niger glucosidase ER GLSI A. nidulans glucosidase ER HDEL Universal in retrieval signal ER at C- fungi terminus SEC12 S. cerevisiae COPII vesicle protein ER/Golgi SEC12 A. niger COPII vesicle protein ER/Golgi OCH1 S. cerevisiae 1,6-mannosyltransferase Golgi (cis) OCH1 P. pastoris 1,6-mannosyltransferase Golgi (cis) MNN9 S. cerevisiae 1,6-mannosyltransferase Golgi complex MNN9 A. niger undetermined Golgi VAN1 S. cerevisiae undetermined Golgi VAN1 A. niger undetermined Golgi ANP1 S. cerevisiae undetermined Golgi HOCI S. cerevisiae undetermined Golgi MNN10 S. cerevisiae undetermined Golgi MNN10 A. niger undetermined Golgi MNN11 S. cerevisiae undetermined Golgi (cis) MNN11 A. niger undetermined Golgi (cis) MNT1 S. cerevisiae 1,2-mannosyltransferase Golgi (cis, medial KTR1 P. pastoris undetermined Golgi (medial) KRE2 P. pastoris Undetermined Golgi (medial) KTR3 P. pastoris Undetermined Golgi (medial) MNN2 S. cerevisiae 1,2-mannosyltransferase Golgi (medial) KTR1 S. cerevisiae Undetermined Golgi (medial) KTR2 S. cerevisiae Undetermined Golgi (medial) MNN1 S. cerevisiae 1,3-mannosyltransferase Golgi (trans) MNN6 S. cerevisiae Phosphomannosyltransfer Golgi (trans) ase 2,6 ST H. sapiens 2,6-sialyltransferase trans Golgi network UDP-Gal T S. pombe UDP-Gal transporter Golgi

In any case, it is highly preferred that targeting peptide sequences are selected which are appropriate for the particular enzymatic activity or activities to function optimally within the sequence of desired glycosylation reactions. For example, in developing a modified microorganism capable of terminal sialylation of nascent N-glycans, a process which occurs in the late Golgi in humans, it is desirable to utilize a sub-library of targeting peptide sequences derived from late Golgi proteins. Similarly, the trimming of Man₈GlcNAc₂ by an α-1,2-mannosidase to give Man₅GlcNAc₂ is an early step in complex N-glycan formation in humans (FIG. 1B). It is therefore desirable to have this reaction occur in the ER or early Golgi of an engineered host microorganism. A sub-library encoding ER and early Golgi retention signals is used.

A series of fusion protein constructs (i.e., a combinatorial DNA library) is then constructed by functionally linking one or a series of targeting peptide sequences to one or a series of sequences encoding catalytic domains. In a preferred embodiment, this is accomplished by the in-frame ligation of a sub-library comprising DNA encoding targeting peptide sequences (above) with a sub-library comprising DNA encoding glycosylation enzymes or catalytically active fragments thereof (see below).

The resulting library comprises synthetic genes encoding targeting peptide sequence-containing fusion proteins. In some cases it is desirable to provide a targeting peptide sequence at the N-terminus of a fusion protein, or in other cases at the C-terminus. In some cases, targeting peptide sequences may be inserted within the open reading frame of an enzyme, provided the protein structure of individual folded domains is not disrupted. Each type of fusion protein is constructed (in a step-wise directed or semi-random fashion) and optimal constructs may be selected upon transformation of host cells and characterization of glycosylation patterns in transformed cells using methods of the invention. Using such methods, lower eukaryotic host cells may be engineered to produce recombinant glycoprotein having desirable N-glucan properties, e.g., tailored to the particular protein of interest.

Generating Additional Sequence Diversity

The method of this embodiment is most effective when a nucleic acid, e.g., a DNA library transformed into the host contains a large diversity of sequences, thereby increasing the probability that at least one transformant will exhibit the desired phenotype. Single amino acid mutations, for example, may drastically alter the activity of glycoprotein processing enzymes (Romero et al., 2000). Accordingly, prior to transformation, a DNA library or a constituent sub-library may be subjected to one or more techniques to generate additional sequence diversity. For example, one or more rounds of gene shuffling, error prone PCR, in vitro mutagenesis or other methods for generating sequence diversity, may be performed to obtain a larger diversity of sequences within the pool of fusion constructs.

Expression Control Sequences

In addition to the open reading frame sequences described above, it is generally preferable to provide each library construct with expression control sequences, such as promoters, transcription terminators, enhancers, ribosome binding sites, and other functional sequences as may be necessary to ensure effective transcription and translation of the fusion proteins upon transformation of fusion constructs into the host organism.

Suitable vector components, e.g., selectable markers, expression control sequences (e.g., promoter, enhancers, terminators and the like) and, optionally, sequences required for autonomous replication in a host cell, are selected as a function of which particular host cell is chosen. Selection criteria for suitable vector components for use in a particular mammalian or a lower eukaryotic host cell are routine. Preferred lower eukaryotic host cells of the invention include but are not limited to: any Pichia sp., including but limited to: Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis and Pichia methanolica; any Saccharomyces sp including but not limited to Saccharomyces cerevisiae; Hansenula polymorpha; any Kluyveromyces sp. including but not limited to Kluyveromyces lactis; Candida albicans; Aspergillus nidulans; Aspergillus niger; Aspergillus oryzae; Trichoderma reseei; Chrysosporium lucknowense; any Fusarium sp. including but not limited to: Fusarium gramineum and Fusarium venenatum; and Neurospora crassa. Where the host is Pichia pastoris, suitable promoters include, for example, the AOX1, AOX2, GAPDH and P40 promoters.

Selectable Markers

It is also preferable to provide each construct with at least one selectable marker, such as a gene to impart drug resistance or to complement a host metabolic lesion. The presence of the marker is useful in the subsequent selection of transformants; for example, in yeast the URA3, HIS4, SUC2, G418, BLA, or SH BLE genes may be used. A multitude of selectable markers are known and available for use in yeast, fungi, plant, insect, mammalian and other eukaryotic host cells. A method for transforming yeast cells by inactivating alternately at least two biosynthetic pathways in methylotrophic yeast is described in US2004/0229306, US2005/0170452, and Nett, 2005, which are hereby incorporated herein by reference. The method involves (a) inactivating a first yeast gene in a pathway involved in synthesizing an amino acid or a nucleotide selected from the group consisting of adenine, arginine, histidine, lysine, methionine, proline and uracil with a first selectable marker thereby rendering the host auxotrophic for the amino acid or nucleotide; and then (b) inactivating a second yeast gene not from the same pathway that was inactivated in (a) involved in synthesizing an amino acid or a nucleotide selected from the group consisting of adenine, arginine, histidine, lysine, methionine, proline and uracil using the yeast gene that was inactivated in (a) as a second selectable marker.

Transformation

The nucleic acid library is then transformed into the host organism. In yeast, any convenient method of DNA transfer may be used, such as electroporation, the lithium chloride method, or the spheroplast method. In filamentous fungi and plant cells, conventional methods include particle bombardment, electroporation and agrobacterium mediated transformation. To produce a stable strain suitable for high-density culture (e.g., fermentation in yeast), it is desirable to integrate the DNA library constructs into the host chromosome. In a preferred embodiment, integration occurs via homologous recombination, using techniques well-known in the art. For example, DNA library elements are provided with flanking sequences homologous to sequences of the host organism. In this manner, integration occurs at a defined site in the host genome, without disruption of desirable or essential genes.

In an especially preferred embodiment, library DNA is integrated into the site of an undesired gene in a host chromosome, effecting the disruption or deletion of the gene. For example, integration into the sites of the OCH1, MNN1, or MNN4 genes allows the expression of the desired library DNA while preventing the expression of enzymes involved in yeast hypermannosylation of glycoproteins. In other embodiments, library DNA may be introduced into the host via a nucleic acid molecule, plasmid, vector (e.g., viral or retroviral vector), chromosome, and may be introduced as an autonomous nucleic acid molecule or by homologous or random integration into the host genome. In any case, it is generally desirable to include with each library DNA construct at least one selectable marker gene to allow ready selection of host organisms that have been stably transformed. Recyclable marker genes such as ura3, which can be selected for or against, are especially suitable.

Screening and Selection Processes

After transformation of the host strain with the DNA library, transformants displaying a desired glycosylation phenotype are selected. Selection may be performed in a single step or by a series of phenotypic enrichment and/or depletion steps using any of a variety of assays or detection methods. Phenotypic characterization may be carried out manually or using automated high-throughput screening equipment. Commonly, a host microorganism displays protein N-glycans on the cell surface, where various glycoproteins are localized.

One may screen for those cells that have the highest concentration of terminal GlcNAc on the cell surface, for example, or for those cells which secrete the protein with the highest terminal GlcNAc content. Such a screen may be based on a visual method, like a staining procedure, the ability to bind specific terminal GlcNAc binding antibodies or lectins conjugated to a marker (such lectins are available from E.Y. Laboratories Inc., San Mateo, Calif.), the reduced ability of specific lectins to bind to terminal mannose residues, the ability to incorporate a radioactively labeled sugar in vitro, altered binding to dyes or charged surfaces, or may be accomplished by using a Fluorescence Assisted Cell Sorting (FACS) device in conjunction with a fluorophore labeled lectin or antibody (Guillen, 1998).

Accordingly, intact cells may be screened for a desired glycosylation phenotype by exposing the cells to a lectin or antibody that binds specifically to the desired N-glycan. A wide variety of oligosaccharide-specific lectins are available commercially (e.g., from EY Laboratories, San Mateo, Calif.). Alternatively, antibodies to specific human or animal N-glycans are available commercially or may be produced using standard techniques. An appropriate lectin or antibody may be conjugated to a reporter molecule, such as a chromophore, fluorophore, radioisotope, or an enzyme having a chromogenic substrate (Guillen, 1998).

Screening may then be performed using analytical methods such as spectrophotometry, fluorimetry, fluorescence activated cell sorting, or scintillation counting. In other cases, it may be necessary to analyze isolated glycoproteins or N-glycans from transformed cells. Protein isolation may be carried out by techniques known in the art. In a preferred embodiment, a reporter protein is secreted into the medium and purified by affinity chromatography (e.g. Ni-affinity or glutathione-S-transferase affinity chromatography). In cases where an isolated N-glycan is preferred, an enzyme such as endo-β-N-acetylglucosaminidase (Genzyme Co., Boston, Mass.; New England Biolabs, Beverly, Mass.) may be used to cleave the N-glycans from glycoproteins. Isolated proteins or N-glycans may then be analyzed by liquid chromatography (e.g. HPLC), mass spectroscopy, or other suitable means. U.S. Pat. No. 5,595,900 teaches several methods by which cells with desired extracellular carbohydrate structures may be identified. In a preferred embodiment, MALDI-TOF mass spectrometry is used to analyze the cleaved N-glycans.

Prior to selection of a desired transformant, it may be desirable to deplete the transformed population of cells having undesired phenotypes. For example, when the method is used to engineer a functional mannosidase activity into cells, the desired transformants will have lower levels of mannose in cellular glycoprotein. Exposing the transformed population to a lethal radioisotope of mannose in the medium depletes the population of transformants having the undesired phenotype, i.e. high levels of incorporated mannose (Huffaker, 1983). Alternatively, a cytotoxic lectin or antibody, directed against an undesirable N-glycan, may be used to deplete a transformed population of undesired phenotypes (e.g., Stanley, 1977). U.S. Pat. No. 5,595,900 teaches several methods by which cells with a desired extracellular carbohydrate structures may be identified. Repeatedly carrying out this strategy allows for the sequential engineering of more and more complex glycans in lower eukaryotes.

To detect host cells having on their surface a high degree of the human-like N-glycan intermediate GlcNAcMan₃GlcNAc₂, for example, one may select for transformants that allow for the most efficient transfer of GlcNAc by GlcNAc Transferase from UDP-GlcNAc in an in vitro cell assay. This screen may be carried out by growing cells harboring the transformed library under selective pressure on an agar plate and transferring individual colonies into a 96-well microliter plate. After growing the cells, the cells are centrifuged, the cells resuspended in buffer, and after addition of UDP-GlcNAc and GnT II, the release of UDP is determined either by HPLC or an enzyme linked assay for UDP. Alternatively, one may use radioactively labeled UDP-GlcNAc and GnT II, wash the cells and then look for the release of radioactive GlcNAc by N-acetylglucosaminidase. All this may be carried manually or automated through the use of high throughput screening equipment. Transformants that release more UDP, in the first assay, or more radioactively labeled GlcNAc in the second assay, are expected to have a higher degree of GlcNAcMan₃GlcNAc₂ on their surface and thus constitute the desired phenotype. Similar assays may be adapted to look at the N-glycans on secreted proteins as well.

Alternatively, one may use any other suitable screen such as a lectin binding assay that is able to reveal altered glycosylation patterns on the surface of transformed cells. In this case the reduced binding of lectins specific to terminal mannoses may be a suitable selection tool. Galantus nivalis lectin binds specifically to terminal α-1,3 mannose, which is expected to be reduced if sufficient mannosidase II activity is present in the Golgi. One may also enrich for desired transformants by carrying out a chromatographic separation step that allows for the removal of cells containing a high terminal mannose content. This separation step would be carried out with a lectin column that specifically binds cells with a high terminal mannose content (e.g., Galantus nivalis lectin bound to agarose, Sigma, St.Louis, Mo.) over those that have a low terminal mannose content.

In addition, one may directly create such fusion protein constructs, as additional information on the localization of active carbohydrate modifying enzymes in different lower eukaryotic hosts becomes available in the scientific literature. For example, it is known that human β1,4-GalTr can be fused to the membrane domain of MNT, a mannosyltransferase from S. cerevisiae, and localized to the Golgi apparatus while retaining its catalytic activity (Schwientek et al., J. Biol. Chem. 270:5483-9 1995). If S. cerevisiae or a related organism is the host to be engineered one may directly incorporate such findings into the overall strategy to obtain complex N-glycans from such a host. Several such gene fragments in P. pastoris have been identified that are related to glycosyltransferases in S. cerevisiae and thus could be used for that purpose.

Alteration of Host Cell Glycosylation Using Fusion Constructs from Combinatorial Libraries

The construction of a preferred combinatorial DNA library is illustrated schematically in FIG. 2 and described in Example 11. The fusion construct may be operably linked to a multitude of vectors, such as expression vectors well-known in the art. A wide variety of such fusion constructs were assembled using representative activities as shown in Table 6. Combinations of targeting peptide/catalytic domains may be assembled for use in targeting glycosyltransferase and glycosidase (such as mannosidase) activities in the ER, Golgi and the trans Golgi network according to the invention. Surprisingly, the same catalytic domain may have no effect to a very profound effect on N-glycosylation patterns, depending on the type of targeting peptide used (see, e.g., Table 7, Example 11).

The present invention also provides for the nucleic acid encoding the fusion constructs described herein, vectors comprising such fusion constructs, and host cells transformed with such fusion constructs.

Mannosidase Fusion Constructs

A representative example of a mannosidase fusion construct derived from a combinatorial DNA library of the invention is pFB8, which a truncated Saccharomyces SEC12 (m) targeting peptide (988-1296 nucleotides of SEC12 from SwissProt P11655) ligated in-frame to a 187 N-terminal amino acid deletion of a mouse α-mannosidase IA (Genbank AN: 6678787). The nomenclature used herein, thus, refers to the targeting peptide/catalytic domain region of a glycosylation enzyme as Saccharomyces SEC12 (m)/mouse mannosidase IA Δ187. The encoded fusion protein localizes in the ER by means of the SEC12 targeting peptide sequence while retaining its mannosidase catalytic domain activity and is capable of producing in vivo N-glycans having a Man₅GlcNAc₂ structure (Example 11; FIG. 6F, FIG. 7B).

The fusion construct pGC5, Saccharomyces MNS1 (m)/mouse mannosidase IB Δ99, is another example of a fusion construct having intracellular mannosidase trimming activity (Example 11; FIG. 5D, FIG. 8B). Fusion construct pBC18-5 (Saccharomyces VAN1 (s)/C. elegans mannosidase IB Δ80) is yet another example of an efficient fusion construct capable of producing N-glycans having a Man₅GlcNAc₂ structure in vivo. By creating a combinatorial DNA library of these and other such mannosidase fusion constructs according to the invention, a skilled artisan may distinguish and select those constructs having optimal intracellular trimming activity from those having relatively low or no activity. Methods using combinatorial DNA libraries of the invention are advantageous because only a select few mannosidase fusion constructs may produce a particularly desired N-glycan in vivo.

In addition, mannosidase trimming activity may be specific to a particular protein of interest. Thus, it is to be further understood that not all targeting peptide/mannosidase catalytic domain fusion constructs may function equally well to produce the proper glycosylation on a glycoprotein of interest. Accordingly, a protein of interest may be introduced into a host cell transfected with a combinatorial DNA library to identify one or more fusion constructs which express a mannosidase activity optimal for the protein of interest. One skilled in the art will be able to produce and select optimal fusion construct(s) using the combinatorial DNA library approach described herein.

It is apparent, moreover, that other such fusion constructs exhibiting localized active mannosidase catalytic domains (or more generally, domains of any enzyme) may be made using techniques such as those exemplified in Example 11 and described herein. It will be a matter of routine experimentation for one skilled in the art to make and use the combinatorial DNA library of the present invention to optimize, for example, Man₅GlcNAc₂ production from a library of fusion constructs in a particular expression vector introduced into a particular host cell.

Glycosyltransferase Fusion Constructs

Similarly, a glycosyltransferase combinatorial DNA library was made using the methods of the invention. A combinatorial DNA library of sequences derived from glycosyltransferase I (GnTI) activities were assembled with targeting peptides and screened for efficient production in a lower eukaryotic host cell of a GlcNAcMan₅GlcNAc₂ N-glycan structure on a marker glycoprotein. A fusion construct shown to produce GlcNAcMan₅GlcNAc₂ (pPB104), Saccharomyces MNN9 (s)/human GnTI Δ38 was identified (Example 15). A wide variety of such GnTI fusion constructs were assembled (Example 15, Table 10). Other combinations of targeting peptide/GnTI catalytic domains can readily be assembled by making a combinatorial DNA library. It is also apparent to one skilled in the art that other such fusion constructs exhibiting glycosyltransferase activity may be made as demonstrated in Example 15. It will be a matter of routine experimentation for one skilled in the art to use the combinatorial DNA library method described herein to optimize GlcNAcMan₅GlcNAc₂ production using a selected fusion construct in a particular expression vector and host cell line.

As stated above for mannosidase fusion constructs, not all targeting peptide/GnTI catalytic domain fusion constructs will function equally well to produce the proper glycosylation on a glycoprotein of interest as described herein. However, one skilled in the art will be able to produce and select optimal fusion construct(s) using a DNA library approach as described herein. Example 15 illustrates a preferred embodiment of a combinatorial DNA library comprising targeting peptides and GnTI catalytic domain fusion constructs involved in producing glycoproteins with predominantly GlcNAcMan₅GlcNAc₂ structure. Example 17 discloses the use of a sialyltransferase fusion construct and two different GnTII and MannII fusion constructs.

Using Multiple Fusion Constructs to Alter Host Cell Glycosylation

In another example of using the methods and libraries of the invention to alter host cell glycosylation, a P. pastoris strain with an OCH1 deletion that expresses a reporter protein (K3) was transformed with multiple fusion constructs isolated from combinatorial libraries of the invention to convert high mannose N-glycans to human-like N-glycans (Example 15). First, the mannosidase fusion construct pFB8 (Saccharomyces SEC12 (m)/mouse mannosidase IA Δ187) was transformed into a P. pastoris strain lacking 1,6 initiating mannosyltransferases activity (i.e. och1 deletion; Example 1). Second, pPB103 comprising a K. lactis MNN2-2 gene (Genbank AN: AF106080) encoding an UDP-GlcNAc transporter was constructed to increase further production of GlcNAcMan₅GlcNAc₂. The addition of the UDP-GlcNAc transporter increased production of GlcNAcMan₅GlcNAc₂ significantly in the P. pastoris strain as illustrated in FIG. 10B. Third, pPB104 comprising Saccharomyces MNN9 (s)/human GnTI Δ38 was introduced into the strain. This P. pastoris strain is referred to as “PBP-3.”

It is understood by one skilled in the art that host cells such as the above-described yeast strains can be sequentially transformed and/or co-transformed with one or more expression vectors. It is also understood that the order of transformation is not particularly relevant in producing the glycoprotein of interest. The skilled artisan recognizes the routine modifications of the procedures disclosed herein may provide improved results in the production of the glycoprotein of interest.

The importance of using a particular targeting peptide sequence with a particular catalytic domain sequence becomes readily apparent from the experiments described herein. The combinatorial DNA library provides a tool for constructing enzyme fusions that are involved in modifying N-glycans on a glycoprotein of interest, which is especially useful in producing human-like glycoproteins. (Any enzyme fusion, however, may be selected using libraries and methods of the invention.) Desired transformants expressing appropriately targeted, active α-1,2-mannosidase produce K3 with N-glycans of the structure Man₅GlcNAc₂ as shown in FIGS. 5D and 5E. This confers a reduced molecular mass to the cleaved glycan compared to the K3 of the parent OCH1 deletion strain, as was detected by MALDI-TOF mass spectrometry in FIG. 5C.

Similarly, the same approach was used to produce another secreted glycoprotein: IFN-β comprising predominantly Man₅GlcNAc₂. The Man₅GlcNAc₂ was removed by PNGase digestion (Papac et al. 1998) and subjected to MALDI-TOF as shown in FIG. 6A-6F. A single prominent peak at 1254 (m/z) confirms Man₅GlcNA₂ production on IFN-β in FIGS. 6E (pGC5) (Saccharomyces MNS1 (m)/mouse mannosidase IB Δ99) and 6F (pFB8) (Saccharomyces SEC12 (m)/mouse mannosidase IA Δ187). Furthermore, in the P. pastoris strain PBP-3 comprising pFB8 (Saccharomyces SEC12 (m)/mouse mannosidase IA Δ187), pPB104 (Saccharomyces MNN9 (s)/human GnTI Δ38) and pPB103 (K. lactis MNN2-2 gene), the hybrid N-glycan GlcNAcMan₅GlcNAc₂ [b] was detected by MALDI-TOF (FIG. 10).

After identifying transformants with a high degree of mannose trimming, additional experiments were performed to confirm that mannosidase (trimming) activity occurred in vivo and was not predominantly the result of extracellular activity in the growth medium (Example 13; FIGS. 7-9).

Sequential Glycosylation Reactions

In a preferred embodiment, such targeting peptide/catalytic domain libraries are designed to incorporate existing information on the sequential nature of glycosylation reactions in higher eukaryotes. Reactions known to occur early in the course of glycoprotein processing require the targeting of enzymes that catalyze such reactions to an early part of the Golgi or the ER. For example, the trimming of Man₈GlcNAc₂ to Man₅GlcNAc₂ by mannosidases is an early step in complex N-glycan formation. Because protein processing is initiated in the ER and then proceeds through the early, medial and late Golgi, it is desirable to have this reaction occur in the ER or early Golgi. When designing a library for mannosidase I localization, for example, one thus attempts to match ER and early Golgi targeting signals with the catalytic domain of mannosidase I. As another example, the sialylation of glycoproteins occurs in Golgi. Thus, when designing a library for expression of sialyltransferase, one thus attempts to match Golgi targeting signals with the catalytic domain of a sialyltransferase.

Codon Optimization and Nucleotide Substitution

The methods of the invention may be performed in conjunction with optimization of the base composition for efficient transcription/translation of the encoded protein in a particular host, such as a fungal host. This includes codon optimization to ensure that the cellular pools of tRNA are sufficient. The foreign genes (ORFS) may contain motifs detrimental to complete transcription/translation in the fungal host and, thus, may require substitution to more amenable sequences. The expression of each introduced protein can be followed both at the transcriptional and translational stages by well known Northern and Western blotting techniques, respectively (Sambrook, J. and Russell, D. W., 2001).

Vectors

In another aspect, the present invention provides vectors (including expression vectors), comprising genes encoding activities glycosylation enzymes, a promoter, a terminator, a selectable marker and targeting flanking regions. Such promoters, terminators, selectable markers and flanking regions are readily available in the art. In a preferred embodiment, the promoter in each case is selected to provide optimal expression of the protein encoded by that particular ORF to allow sufficient catalysis of the desired enzymatic reaction. This step requires choosing a promoter that is either constitutive or inducible, and provides regulated levels of transcription. In another embodiment, the terminator selected enables sufficient termination of transcription. In yet another embodiment, the selectable markers used are unique to each ORF to enable the subsequent selection of a fungal strain that contains a specific combination of the ORFs to be introduced. In a further embodiment, the locus to which each fusion construct (encoding promoter, ORF and terminator) is localized, is determined by the choice of flanking region. The present invention is not limited to the use of the vectors disclosed herein.

Integration Sites

As one ultimate goal of this genetic engineering effort is a robust protein production strain that is able to perform well in an industrial fermentation process, the integration of multiple genes into the host (e.g., fungal) chromosome preferably involves careful planning. The engineered strain may likely have to be transformed with a range of different genes, and these genes will have to be transformed in a stable fashion to ensure that the desired activity is maintained throughout the fermentation process. As described herein, any combination of various desired enzyme activities may be engineered into the fungal protein expression host, e.g., sialyltransferases, mannosidases, fucosyltransferases, galactosyltransferases, glucosyltransferases, GlcNAc transferases, ER and Golgi specific transporters (e.g. syn and antiport transporters for UDP-galactose and other precursors), other enzymes involved in the processing of oligosaccharides, and enzymes involved in the synthesis of activated oligosaccharide precursors such as UDP-galactose, CMP-N-acetylneuraminic acid. Genes which encode enzymes known to be characteristic of non-human glycosylation reactions in fugal hosts and their corresponding proteins have been extensively characterized in a number of lower eukaryotes (e.g., Saccharomyces cerevisiae, Trichoderma reesei, Aspergillus nidulans, P. pastoris, etc.), thereby providing a list of known glycosyltransferases in lower eukaryotes, their activities and their respective genetic sequence. These genes are likely to be selected from the group of mannosyltransferases e.g., 1,3 mannosyltransferases (e.g., MNN1 in S. cerevisiae) (Graham, 1991), 1,2 mannosyltransferases (e.g., the KTR/KRE family from S. cerevisiae), 1,6 mannosyltransferases (OCH1 from S. cerevisiae), mannosylphosphate transferases and their regulators (MNN4 and MNN6 from S. cerevisiae) and additional enzymes that are involved in aberrant (i.e. non-human) glycosylation reactions. Examples of preferred methods for modifying glycosylation in a lower eukaryotic host cell, such as Pichia pastoris, are shown in Table 6. (The HDEL and KDEL signal peptides in the second row of the third column are shown in SEQ ID NOS: 5 and 6, respectively).

TABLE 6. Some Preferred Embodiments For Modifying Glycosylation In A Lower Eukaroytic Microorganism Suitable Suitable Sources Suitable Transporters Desired Suitable Catalytic of Localization Gene and/or Structure Activities Sequences Deletions Phosphatases Man₅GlcNAc₂ a-1,2-mannosidase Mns1 OCH1 none (murine, human, (N-terminus, MNN4 Bacillus sp., S. cerevisiae) MNN6 A. nidulans) Och1 (N-terminus, S. cerevisiae, P. pastoris) Ktr1 Mnn9 Mnt1 (S. cerevisiae) KDEL, HDEL (C-terminus) GlcNAcMan₅ GlcNAc Och1 OCH1 UDP-GlcNAc GlcNAc₂ Transferase I, (N-terminus, MNN4 transporter (human, murine, rat S. cerevisiae, MNN6 (human, etc.) P. pastoris) murine, KTR1 K. lactis) (N-terminus) UDPase Mnn1 (human) (N-terminus, S. cerevisiae) Mnt1 (N-terminus, S. cerevisiae) GDPase (N-terminus, S. cerevisiae) GlcNAcMan₃ mannosidase II Ktrl OCH1 UDP-GlcNAc GlcNAc₂ Mnn1 MNN4 transporter (N-terminus, MNN6 (human, S. cerevisiae) murine, Mnt1 (N-terminus, K. lactis) S. cerevisiae) UDPase Kre2/Mnt1 (human) (S. cerevisiae) Kre2 (P. pastoris) Ktr1 (S. cerevisiae) Ktr1 (P. pastoris) Mnn1 (S. cerevisiae) GlcNAc₍₂₋₄₎ GlcNAc Mnn1 OCH1 UDP-GlcNAc Man₃GlcNAc₂ Transferase II, III, (N-terminus, MNN4 transporter IV, V (human, S. cerevisiae) MNN6 (human, murine) Mnt1 murine, (N-terminus, K. lactis) S. cerevisiae) UDPase Kre2/Mnt1 (human) (S. cerevisiae) Kre2 (P. pastoris) Ktr1 (S. cerevisiae) Ktr1 (P. pastoris) Mnn1 (S. cerevisiae) Gal₍₁₋₄₎ β-1,4-Galactosyl Mnn1 OCH1 UDP-Galactose GlcNAc₍₂₋₄₎- transferase (human) (N-terminus, MNN4 transporter Man₃GlcNAc₂ S. cerevisiae) MNN6 (human, Mnt1(N-terminus, S. pombe) S. cerevisiae) Kre2/Mnt 1 (S. cerevisiae) Kre2 (P. pastoris) Ktr1 (S. cerevisiae) Ktrl (P.pastoris) Mnn1 (S. cerevisiae) NANA₍₁₋₄₎- α-2,6- KTR1 OCH1 CMP-Sialic Gal₍₁₋₄₎ Sialyltransferase MNN1 MNN4 acid G1cNAc₍₂₋₄₎- (human) (N-terminus, MNN6 transporter Man₃GlcNAc₂ α-2,3- S. cerevisiae) (human) Sialyltransferase MNT1 (N-terminus, S. cerevisiae) Kre2/Mnt1 (S. cerevisiae) Kre2 (P. pastoris) Ktr1 (S. cerevisiae) Ktr1 (P. pastoris) MNN1 (S. cerevisiae) MNN2 (S. cerevisiae)

Methods for Producing CMP-Sia for the Generation of Recombinant N-Glycans

The present invention provides methods for production of a functional CMP-Sia biosynthetic pathway in a host cell that lacks endogenous CMP-Sia, such as a fungal cell. The present invention also provides a method for creating a host cell that has been modified to express a new or altered CMP-Sia pathway. The invention further provides a method for creating a host cell that comprises a cellular pool of CMP-Sia.

The methods involve the cloning and expression in a host cell of several genes encoding enzymes of the CMP-Sia biosynthetic pathway resulting in a cellular pool of CMP-Sia in the host cell which can be utilized in the production of sialylated glycans on proteins of interest. In general, the addition of sialic acids to glycans requires the presence of the sialyltransferase, a glycan acceptor (e.g., Gal₂GlcNAc₂Man₃GlcNAc₂) and the sialyl donor molecule, CMP-Sia. The synthesis of the CMP-Sia donor molecule in higher organisms (e.g., mammals) is a four enzyme, multiple reaction process starting with the substrate UDP-GlcNAc and resulting in CMP-Sia (FIG. 14A). The process initiates in the cytoplasm producing sialic acid which is then translocated into the nucleus where Sia is converted to CMP-Sia by CMP-sialic acid synthase. Subsequently, CMP-Sia exits the nucleus into the cytoplasm and is then transported into the Golgi where sialyltransferases catalyze the transfer of sialic acid onto the acceptor glycan. In contrast, the bacterial pathway for synthesizing CMP-Sia from UDP-GlcNAc involves only three enzymes and two intermediates (FIG. 14), with all reactions occurring in the cytoplasm.

Accordingly, the methods of the invention involve generating a pool of CMP-Sia in a non-human host cell that lacks endogenous CMP-Sia by introducing a functional CMP-Sia biosynthetic pathway. With readily available DNA sequence information from genetic databases (e.g., GenBank, Swissprot), enzymes and/or activities involved in the CMP-Sia pathways (Example 16) are cloned. Using standard techniques known to those skilled in the art, nucleic acid molecules encoding one or more enzymes (or catalytically active fragments thereof) involved in the biosynthesis or transport of CMP-Sia are inserted into appropriate expression vectors under the transcriptional control of promoters and/or other expression control sequences capable of driving transcription in a selected host cell of the invention (e.g., a fungal host cell). The functional expression of such enzymes in the selected host cells of the invention can be detected. In one embodiment, the functional expression of such enzymes in the selected host cells of the invention can be detected by measuring the intermediate formed by the enzyme. The methods of the invention are not limited to the use of the specific enzyme sources disclosed herein. In certain preferred embodiments, combinatorial libraries of the invention are utilized to select and/or optimize CMP-Sia pathway activity in the selected host cell.

Engineering a Mammalian CMP-Sialic Acid Biosynthetic Pathway in a Host Cell

In one embodiment of the invention, the method involves cloning several genes encoding enzymes in the CMP-Sia biosynthetic pathway, including UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase, N-acetylneuraminate-9-phosphate synthase, N-acetylneuraminate-9-phosphatase, CMP-sialic acid phosphatase and CMP-sialic acid synthase, and expressing one or more of said cloned genes in a host cell which lacks endogenous CMP-Sia, such as a fungal host cell. The genes are expressed to generate each enzyme, producing intermediates that are used for subsequent enzymatic reactions. Example 16 describe methods for the introduction of these enzymes into a fungal host (e.g., P. pastoris) using a selection marker. Alternatively, the enzymes are expressed together to produce or increase downstream intermediates whereby subsequent enzymes are able to act upon them.

The first enzyme in the pathway is a bi-functional enzyme that is both a UDP-GlcNAc epimerase and an N-acetylmannosamine kinase, converting UDP-GlcNAc through N-acetylmannosamine (ManNAc) to N-acetylmannosamine-6-phosphate (ManNAc-6-P) (Hinderlich, 1997). This enzyme was originally cloned from a rat liver cDNA library (Stasche, 1997). Homologs to this enzyme have subsequently been found to exist in other species, for example, human (NM_(—)005476); E. coli (Ringenberg et al. 2003); Mannheimia haemolytica (McKerrell and Lo, Infect Immun. 2002 May; 70(5):2622-9). In a preferred embodiment, a gene encoding the functional UDP-N-acetylglucosamine-2-epimerase enzyme, including homologs, variants and derivatives thereof, is cloned and expressed in a host cell (for example, a non-human host cell which lacks endogenous CMP-Sia, such as a fungal host cell). In another preferred embodiment, a gene encoding the functional N-acetylmannosamine kinase enzyme, including homologs, variants and derivatives thereof, is cloned and expressed in a host cell, such as a fungal host cell. In a more preferred embodiment, a gene encoding the bifunctional UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase enzyme, including homologs, variants and derivatives thereof, is cloned and expressed in a host cell, such as a fungal host cell (e.g., P. pastoris). The functional expression of these genes can be detected using a functional assay. In one embodiment, the functional expression of such genes can be detected by monitoring the formation of ManNAc and ManNAc-6-P intermediates.

The second enzyme in the pathway, N-acetylneuraminic acid phosphate synthase, was cloned from human liver based on its homology to the E. coli sialic acid synthase gene, NeuB (Lawrence, 2000). Homologs of the N-acetylneuraminic acid phosphate synthase enzyme are also known in rat liver (Chen et al., Glycobiology. 2002 12(2):65-71) and mouse (Nakata et al., Biochem. Biophys. Res. Commun. 2000; 273(2):642-8). This enzyme catalyzes the conversion of ManNAc-6-P to sialate 9-phosphate (also referred to as Sia-9P, N-acetylneuraminate 9-phosphate, or Neu5Ac-9P). Accordingly, in a preferred embodiment, a gene encoding the functional N-acetylneuraminate 9-phosphate synthase enzyme, including homologs, variants and derivatives thereof, is cloned and expressed in a host cell (for example a non-human host cell which lacks endogenous CMP-Sia, such as a fungal host cell). The expression of N-acetyl-neuraminic acid phosphate synthase in the host can be detected using a functional assay. In one embodiment, the functional expression of N-acetylneuraminic acid phosphate synthase can be detected by monitoring the formation of Sia-9P.

The third enzyme in the pathway, N-acetylneuraminate 9-phosphatase (Sia-9-phosphatase), is involved in the conversion of Sia-9-P to sialic acid. The cloning of this enzyme has recently been reported by Maliekal et al., 2006 (See GenBank AN NM_(—)152667). Although the activity of this enzyme has been detected in mammalian cells, no such activity has been identified in fungal cells. Therefore, the lack of Sia-9-phosphatase would cause a break in the pathway. Accordingly, in a preferred embodiment, the method of the present invention involves isolating and cloning a Sia-9-phosphatase gene and expressing it in a non-human host cell, such as a fungal host cell. (See Example 16.) Such hosts include, e.g., yeast, fungal, insect and bacterial cells. In a more preferred embodiment, the Sia-9-phosphatase gene, including homologs, variants and derivatives thereof, is expressed in a non-human host cell that lacks endogenous CMP-Sia or that expresses inadequate levels of CMP-Sia, such as a fungal host. The expression of Sia-9-phosphatase in the host can be detected using a functional assay. In one embodiment, the functional expression of Sia-9-phosphatase can be detected by monitoring the formation of sialic acid.

The next enzyme in the mammalian pathway, CMP-Sia synthase, was originally cloned from the murine pituitary gland by functional complementation of a cell line deficient in this enzyme (Munster, 1998). Homologs have been found to exist in E. coli (Mercker and Troy, 1990); and human (Raju et al., 2001). This enzyme converts sialic acid to CMP-Sia, which is the donor substrate in a sialyl-transferase reaction in the Golgi. Accordingly, in an even more preferred embodiment, a gene encoding the functional CMP-Sia synthase enzyme, including homologs, variants and derivatives thereof, is cloned and expressed in a host cell (for example a non-human that lacks endogenous CMP-Sia, such as a fungal host cell). The expression of CMP-Sia synthase in the host can be detected using a functional assay. In one embodiment, the functional expression of CMP-Sia synthase can be detected by monitoring the formation of CMP-Sia.

The method of the present invention further involves the production of the intermediates produced in a host cell as a result of expressing the above enzymes in the CMP-Sia pathway. Preferably, the intermediates produced include one or more of the following: UDP-GlcNAc, ManNAc, ManNAc-6-P, Sia-9-P, Sia and CMP-Sia. Additionally, each intermediate produced by the enzymes is preferably detected. For example, to detect the presence or absence of an intermediate, an assay as described in Example 16 is used. Accordingly, the invention also provides assays to detect the N-glycan intermediates produced in a non-human host cell that lacks endogenous CMP-Sia, such as a fungal host cell.

A skilled artisan recognizes that the mere availability of one or more enzymes in the CMP-sialic acid biosynthetic pathway does not suggest that such enzymes can be functionally expressed in a host cell that lacks endogenous CMP-Sia, such as a fungal host cell. To date, the ability of such host cell to express these mammalian enzymes to create a functional de novo CMP-Sia biosynthetic pathway has not been described. The present invention provides for the first time the functional expression of at least one mammalian enzyme involved in CMP-Sia biosynthesis in a fungal host: the mouse CMP-Sia synthase (Example 16), indicating that production of CMP-Sia via the mammalian pathway (in whole or in part) is possible in a fungal host and thus in other non-human hosts that lack endogenous CMP-Sia.

The invention described herein is not limited to the use of the specific enzymes, genes, plasmids and constructs disclosed herein. A person of skill would readily understand how to use any homologs, variants, derivatives and functional equivalents of the genes involved in the synthesis of CMP-Sia.

To produce sialylated, recombinant glycoproteins in a host cell that lacks endogenous CMP-Sia (e.g., a fungal host such as P. pastoris) or in a host cell that needs or can benefit from increased levels of CMP-Sia, the above mentioned mammalian enzymes can be expressed using a combinatorial DNA library approach as disclosed herein, generating a pool of CMP-Sia, which is transferred onto galactosylated N-glycans in the presence of a sialyltransferase. Accordingly, the present invention provides a method for engineering a CMP-Sia biosynthetic pathway into a host cell by expressing one or more of each of the enzymes such that they function, preferably so that they function optimally, in the subcellular location to which they are targeted in the selected host cell. Mammalian, bacterial or hybrid engineered CMP-Sia biosynthetic pathways are provided.

Engineering a Bacterial CMP-Sialic Acid Biosynthetic Pathway in a Host Cell

The metabolic intermediate UDP-GlcNAc is common to eukaryotes and prokaryotes, providing an endogenous substrate from which to initiate the synthesis of CMP-Sia (FIG. 14). Based on the presence of this common intermediate, the CMP-Sia biosynthetic pathway can be engineered into non-human host cells that lack endogenous CMP-Sia by introducing the genes encoding the bacterial UDP-GlcNAc epimerase (NeuC), sialate synthase (NeuB) and CMP-Sia (NeuA) synthase into the host cell, e.g., by transformation methods such as on plasmids, and preferably, by integration into the host cell chromosome. Accordingly, another aspect of the present invention involves engineering a bacterial CMP-Sia biosynthetic pathway into host cells that lack an endogenous CMP-Sia pathway or into host cells that need or would benefit from increased levels of CMP-Sia. The expression of bacterial Neu genes in cells that lack an endogenous CMP-Sia biosynthetic pathway enables the generation of a cellular CMP-Sia pool, which can subsequently facilitate the production of recombinant N-glycans having detectable level of sialylation on a protein of interest, such as recombinantly expressed glycoproteins. The bacterial enzymes involved in the synthesis of CMP-Sia include UDP-GlcNAc epimerase (NeuC), sialate synthase (NeuB) and CMP-Sia synthase (NeuA). In one embodiment, the NeuC, NeuB, and NeuA genes, which encode these functional enzymes, respectively, including homologs, variants and derivatives thereof, are cloned and expressed in non-human host cells that lack an endogenous CMP-Sia pathway, such as a fungal host cell. The sequences of NeuC, NeuB and NeuA genes are shown in FIGS. 15-17, respectively. The expression of these genes in the host cell generates the intermediate molecules in the biosynthetic pathway of CMP-sialic acid (FIG. 14B).

In addition to these three enzymes, the method for synthesizing the bacterial CMP-Sia biosynthetic pathway from UDP-GlcNAc involves generating two intermediates: ManNAc and Sia (FIG. 14B). The conversion of UDP-GlcNAc to ManNAc is facilitated by the NeuC gene. The conversion of ManNAc to Sia is facilitated by the NeuB gene and the conversion of Sia to CMP-Sia is facilitated by the NeuA gene. These three enzymes (or homologs thereof) have thus far been found linked together in pathogenic bacteria—i.e., not single gene has been found without the other two. Introducing the bacterial pathway into a host cell, such as a fungal host, thus requires the manipulation of fewer genes than introducing mammalian pathway.

The E. coli UDP-GlcNAc epimerase, encoded by the E. coli NeuC gene, is the first enzyme involved in the bacterial synthesis of polysialic acid (Ringenberg, 2001). The NeuC gene (Genbank AN: M84026.1; SEQ ID NO:57) encoding this enzyme was isolated from the pathogenic E. coli K1 strain and encodes a protein of 391 amino acids (SEQ ID NO:58) (FIG. 15) (Zapata, 1992). The encoded UDP-GlcNAc epimerase catalyzes the conversion of UDP-GlcNAc to ManNAc. Homologs of this enzyme have been identified in several pathogenic bacteria, including Streptococcus agalactiae, Synechococcus sp. WH 8102, Clostridium thermocellum, Vibrio vulnificus, Legionella pnuemophila, and Campylobacter jejuni. In one embodiment, a gene encoding the functional E. coli UDP-GlcNAc epimerase enzyme (NeuC), including homologs, variants and derivatives thereof, is cloned and expressed in a host cell (for example a non-human host cell, such as a fungal host). The expression of NeuC in the host can be detected using a functional assay. In one embodiment, the functional expression NeuC can be detected by monitoring the formation of ManNAc.

The second enzyme in the bacterial pathway is sialate synthase, which directly converts ManNAc to Sia, bypassing several, enzymes and intermediates present in the mammalian pathway. This enzyme of 346 amino acids (SEQ ID NO:60), is encoded by the E. coli NeuB gene (Genbank AN: U05248.1; SEQ ID NO:59) (FIG. 16) (Annunziato, 1995). In another embodiment, a gene encoding a functional E. coli sialate synthase enzyme (NeuB), including homologs, variants and derivatives thereof, is cloned and expressed in a host cell (for example, a non-human host cell, such as a fungal host cell). The expression of NeuB in the host can be detected using a functional assay. In one embodiment, the functional expression NeuB can be detected by monitoring the formation of Sia.

The third enzyme in this bacterial pathway is CMP-Sia synthase, consisting of 419 amino acids (SEQ ID NO:62) and encoded by the E. coli NeuA gene (Genbank AN: J05023; SEQ ID NO:61) (FIG. 17). CMP-Sia synthase converts Sia to CMP-Sia (Zapata, 1989). The NeuA gene is found in the same organisms as the NeuC and NeuB genes. Accordingly, in yet another embodiment, a gene (NeuA) encoding a functional E. coli CMP-Sia synthase enzyme, including homologs, variants and derivatives thereof, is cloned and expressed in a host cell (for example, a non-human host cell, such as a fungal host cell). In one embodiment, the expression NeuA can be detected by monitoring the formation of CMP-Sia.

In yet another embodiment, the gene encoding a functional bacterial CMP-Sia synthase (e.g. NeuA) encodes a fusion protein comprising a: catalytic domain having the activity of a bacterial CMP-Sia synthase and a cellular targeting signal peptide (not normally associated with the catalytic domain) selected to target the enzyme to the nucleus of the host cell. In one embodiment, said cellular targeting signal peptide comprises a domain of the SV40 capside polypeptide VP1. In another embodiment, the signal peptide comprises one or more endogenous signaling motifs from a mammalian CMP-Sia synthase that ensure correct localization of the enzyme to the nucleus. Methods for making said fusion protein are well known in the art.

After PCR amplification of the E. coli NeuA, NeuB and NeuC genes, the amplified fragments were ligated into a selectable yeast integration vector under the control of a promoter (Example 16). After transforming a host strain (e.g., P. pastoris), with each vector carrying the Neu gene fragments, colonies were screened by applying positive selection for the presence of vectors and then screening transformants for Neu gene enzymatic activity. The ability of a non-human host cell that lacks endogenous sialylation to express the bacterial enzymes involved in creating a de novo CMP-Sia biosynthetic pathway is provided for the first time by this invention.

Engineering a Hybrid Mammalian/Bacterial CMP-Sialic Acid Biosynthetic Pathway in a Host Cell

Both mammalian and bacterial CMP-Sia biosynthetic pathways require that both CTP and sialic acid be available to the CMP-Sia synthase. Although similar in enzymatic function to the corresponding bacterial enzyme, the mammalian CMP-Sia synthase may include one or more endogenous signaling motifs that ensure correct localization to the nucleus. Because eukaryotes have a nucleus-localized pool of CTP and the prokaryotic CMP-Sia synthase may not localize to this compartment, a hybrid CMP-Sia biosynthetic pathway combining both mammalian and bacterial enzymes is a preferred method for the production of sialic acid and its intermediates in a non-human host cell that lacks endogenous sialylation, such as a fungal host cell. To this end, a pathway can be engineered into the host cell which involves the integration of both NeuC and NeuB as well as a mammalian CMP-Sia synthase. The CMP-Sia synthase enzyme may be selected from several mammalian homologs that have been cloned and characterized (Genbank AN: AJ006215; SEQ ID NO:63) (Munster, 1998) (see e.g., the murine CMP-Sia synthase) (FIG. 18). In one preferred embodiment, the host cell is transformed with UDP-GlcNAc epimerase (E. coli NeuC) and sialate synthase (E. coli NeuB) in combination with the mouse CMP-Sia synthase. The host engineered with this hybrid CMP-Sia biosynthetic pathway produces a cellular pool of the donor molecule CMP-Sia (FIG. 25). In a more preferred embodiment, the combination of the enzymes expressed in the host is selected for enhanced production of the donor molecule CMP-Sia.

Engineering Enzymes Involved in Alternative Routes for Enhancing the Production of CMP-Sialic Acid Pathway Intermediates in Host Cells

In yet another aspect of the invention, enzymes involved in alternate pathways of CMP-sialic acid biosynthesis are engineered into non-human host cells that lack endogenous sialylation, such as fungal host cells; or in host cells in which endogenous sialylation may be altered, e.g., enhanced. For example, it is contemplated that when an intermediate becomes limiting during one of the methods outlined above, the introduction of an enzyme that uses an alternate mechanism to produce that intermediate will serve as a sufficient substitute in the production of CMP-sialic acid, or any intermediate along this pathway. Embodiments are described herein for the production of the intermediates ManNAc and Sia, though this approach may be extended to produce other intermediates. Furthermore, any of these enzymes can be incorporated into either the mammalian, bacterial or hybrid pathways, either in the absence of the enzymes mentioned previously (i.e., enzymes producing the same intermediate) or in the presence of enzymes mentioned previously, i.e., to enhance overall production.

In the above mentioned embodiments, ManNAc is produced from UDP-GlcNAc by either the mammalian enzyme UDP-GlcNAc-2-epimerase/ManNAc kinase or by the bacterial enzyme NeuC. The substrate for this reaction, UDP-GlcNAc, is predicted to be present in sufficient quantities in cells for the synthesis of CMP-Sia due to its requirement in producing several classes of molecules, including endogenous N-glycans. However, if ManNAc does become limiting—potentially due to the increased demand for ManNAc from the sialic acid biosynthetic pathway—then the cellular supply of ManNAc may be increased by introducing a GlcNAc epimerase which reacts with the substrate GlcNAc to produce ManNAc.

Accordingly, in one embodiment, a gene encoding a functional GlcNAc epimerase enzyme, including homologs, variants and derivatives thereof, is cloned and expressed in a host cell. Using GlcNAc epimerase to directly convert GlcNAc to ManNAc is a shorter, more efficient approach compared with the two-step process involving the synthesis of UDP-GlcNAc (FIG. 19). The GlcNAc epimerase is readily available and, to date, the only confirmed GlcNAc epimerase to have been cloned is from the pig kidney (Maru, 1996) (Example 16). The gene (Genbank AN: D83766; SEQ ID NO: 65) isolated from pig kidney encodes a protein of 402 amino acids (SEQ ID NO: 64) (FIG. 20). When this enzyme was cloned, it was found to be identical to the pig renin-binding protein cloned previously (Inoue, 1990). Although this is the only protein with confirmed GlcNAc epimerase activity, several other renin-binding proteins have been isolated from other organisms, including humans, mouse, rat and bacteria, among others. All are shown to have significant homology. For example, the human GlcNAc epimerase homolog (Genbank AN: D10232.1) has 87% identity and 92% similarity to the pig GlcNAc epimerase protein. Although these homologs are very similar in sequence, the pig protein is the only one having demonstrable epimerase activity to date. The methods of the invention may be performed using any gene encoding a functional GlcNAc epimerase activity. Based on the presence of GlcNAc epimerase activity, the cloning and expression of this gene in a non-human host cell, such as a fungal host cell, is predicted to enhance the cellular levels of ManNAc, thereby, providing sufficient substrate for the enzymes that utilize ManNAc in the CMP-sialic acid biosynthetic pathway.

In another embodiment, sialate aldolase is used to increase cellular levels of sialic acid, as illustrated in FIG. 21. This enzyme (also known as sialate lyase and sialate pyruvate-lyase) directly catalyzes the reversible reaction of ManNAc to sialic acid. In the presence of low concentrations of Sia, this enzyme catalyzes the condensation of ManNAc and pyruvate to produce Sia. Conversely, when Sia concentrations are high, the enzyme causes the reverse reaction to proceed, producing ManNAc and pyruvate (Vimr, 1985). In the above embodiments, the presence of CMP-Sia synthase converts substantially all Sia to CMP-Sia, thus shifting the equilibrium of the aldolase reaction to the condensation of ManNAc and pyruvate to produce Sia. Preferably, the sialate aldolase used in this embodiment is expressed from the E. coli NanA gene, but the invention is not limited to this enzyme source. The gene (Genbank AN: X03345; SEQ ID NO:67) for this enzyme encodes a 297 amino acid protein (SEQ ID NO:68) (FIG. 22) (Ohta, 1985). Close homologs to this enzyme are found in many pathogenic bacteria, including, Salmonella typhimurium, Staphylococcus aureus, Clostridium perfringens, Haemophilus influenzae among others. In addition, homologs are also present in mammals, including mice and humans. In one embodiment, cloning a gene encoding a sialate aldolase activity and expressing it in a fungal host cell enhances the cellular levels of Sia, thereby providing sufficient substrate for the enzymes that utilize Sia in the CMP-sialic acid biosynthetic pathway (Example 16).

Regulation of CMP-Sialic Acid Synthesis: Feedback Inhibition and Inducible Promoters

In mammalian cells, the production of CMP-sialic acid is highly regulated. CMP-sialic acid acts as a feedback inhibitor, acting on UDP-GlcNAc epimerase/ManNAc kinase to prevent further production of CMP-Sia (Hinderlich, 1997; Keppler, 1999). In contrast, the bacterial CMP-Sia biosynthetic pathway (FIG. 14B) does not appear to have a feedback inhibitory control mechanism that would limit the production of CMP-Sia (Ringenberg, 2001). However, incorporation of the E. coli sialate aldolase into one of the pathways mentioned above could cause a shift in the direction of the reaction that it catalyzes, depending on the balance of the equilibrium, thus potentially causing hydrolysis of Sia back to ManNAc. Accordingly, the methods involving sialate aldolase as outlined above will prevent this reverse reaction from occurring, given the presence of CMP-sialate synthase, which rapidly converts Sia to CMP-Sia.

The embodiments described thus far have detailed the constitutive over-expression of the enzymes in a particular biosynthetic pathway of CMP-Sia. Though no literature is currently available that suggests that the presence of any of the mentioned intermediates, and/or the final product could be detrimental to a non-human host, such as a fungal host, a preferred embodiment of the invention has one or more of the enzymes under the control of a regulatable (e.g., an inducible) promoter. In this embodiment, the gene (or ORF) encoding the protein of interest (including but not limited to: UDP-GlcNAc 2-epimerase/ManNAc kinase, NeuC, and GlcNAc epimerase) is cloned downstream of an inducible or regulatable promoter (including but not limited to: the alcohol oxidase promoter (AOX1 or AOX2; Tschopp, 1987), galactose-inducible promoter (GAL10; Yocum, 1984), tetracycline-inducible promoter (TET; Belli, 1998)) to facilitate the controlled expression of that enzyme, and thus to regulate the production of CMP-Sia.

Detection of CMP-Sialic Acid and the Intermediate Compounds in its Synthesis

The methods of the present invention provide engineered pathways to produce a cellular pool of CMP-Sia in non-human host cells that lack an endogenous CMP-Sia biosynthetic pathway, and/or to enhance production of CMP-Sia in host cells that have an endogenous pathway. To assess the production of each intermediate in the pathway, these intermediates must be detectable. Accordingly, the present invention also provides a method for detecting such intermediates. A method for detecting a cellular pool of CMP-Sia, for example, is provided in Example 16). Currently, the literature describes only a few methods for measuring cellular CMP-Sia and its precursors. Early methods involved paper chromatography and thiobarbituric acid analysis and were found to be complicated and time consuming (Briles, 1977; Harms, 1973). HPLC (high pressure liquid chromatography) has also been used, though earlier methods employed acid elution resulting in the rapid hydrolysis of the CMP-Sia (Rump, 1986). Most recently, a more robust method has been described using high-performance anion-exchange chromatography using an alkaline elution protocol combined with pulsed amperometric detection (HPAEC-PAD) (Fritsch, 1996). This method, in addition to detecting CMP-Sia, can also detect the precursor sialic acid, thus being useful for confirming cellular synthesis of either or both of these compounds.

Methods for in Vivo Transfer of Sialic Acid to N-Glycans Using Sialyltransferase

The present invention provides a method for producing a sialylated glycoprotein in a recombinant non-human eukaryotic host cell. In one embodiment, the non-human eukaryotic host cell does not normally display sialyltransferase activity.

In one embodiment, said method comprises introducing into the host a nucleic acid encoding a sialyltransferase enzyme. The enzyme having sialyltransferase activity can be any enzyme having sialyltransferase activity including, but not limited to, any α-2,3 ST, any α-2,6 ST, human ST6Gal (GenBank AN: NM_(—)00302) or a homologue thereof, human ST3Gal (GenBank AN: L23767) or a homologue thereof, or Xenopus ST3Gal (GenBank AN: CAF22058) or a homologue thereof.

As used herein, a homologue of human ST6Gal refers to a nucleic acid having significant alignment with human ST6Gal (NM_(—)003032). Homologues of human ST6Gal include the enzymes identified by GenBank Accession Nos.: AAH40009.1, CAF29492.1, CAI29584.1, CAA38246.1, CAA75385.1, AAH92222.1, P13721, Q64685, NP_(—)990572.1, XP_(—)535839.1, XP_(—)517005.1, NP_(—)775324.1, XP_(—)516938.1, NP_(—)001003853.1, CAI39643.1, CAI29183.1, CAG32836.1, XP_(—)545243.1, CAF29496.1, XP_(—)416927.1, CAF29497.1, AAB22858.1, NP_(—)671738.1, CAI29184.1, AAB22859.1, BAC24793.1, BAB47506.1, CAF29493.1, XP_(—)515705.1, XP_(—)614392.1, CAF29495.1, XP_(—)236826.2, BAC87752.1, CAI29185.1, CAF97336.1, CAI39644.1, CAD54408.1, CAG05808.1, XP_(—)538436.1, BAC98272.1, XP_(—)604448.1, AAD33059.1, BAC28828.1, BAB00636.1, AAH08680.1, NP_(—)766417.1, EAA04038.2, CAH25390.1, NP_(—)726474.1, and NP_(—)523853.1.

As used herein, a homologue of human ST3Gal refers to a nucleic acid having significant alignment with human ST3Gal (L23767). Homologues of human ST3Gal include the enzymes identified by GenBank Accession Nos.: AAA16460.1, AAM81378.1, AAH10645.1, CAH90316.1, AAM66433.1, CAF25182.1, AAK93790.1, AAM66431.1, CAA52662.1, AAM66432.1, AAF28871.1, NP_(—)976082.1, AAH11121.1, NP_(—)033204.2, AAP22942.1, CAA65076.1, NP_(—)998922.1, NP_(—)991375.1, CAB53395.1, XP_(—)522245.1, AAC14162.1, CAI29182.1, XP_(—)417860.1, AAC14163.1, BAB25732.1, BAB13940.1, CAF22058.1, NP_(—)001003854.1, CAG00953.1, CAI26289.1, XP_(—)546408.1, YP_(—)227525.1, AAH84840.1, CAF25054.1, NP_(—)989810.1, CAF25503.1, Q6 KB54, NP_(—)001002883.1, AAH23312.1, CAH89922.1, CAF25178.1, AAH53179.1, AA013870.1, AA013869.1, AA013867.1, AA013866.1, AA013861.1, AA013859.1, NP_(—)777631.1, and NP_(—)777628.1.

As used herein, a homologue of Xenopus ST3Gal refers to a nucleic acid having significant alignment with Xenopus ST3Gal (CAF22058). Homologues of Xenopus ST3Gal include the enzymes identified by GenBank Accession Nos.: CAF22058.1, CAI29182.1, XP_(—)417860.1, AAH10645.1, AAA16460.1, AAF28871.1, CAH90316.1, CAA52662.1, AAM66433.1, AAM66432.1, AAH11121.1, NP_(—)976082.1, AAP22942.1, NP_(—)033204.2, NP_(—)998922.1, CAF25182.1, NP_(—)991375.1, AAK93790.1, AAM81378.1, CAA65076.1, AAM66431.1, CAB53395.1, AAC14162.1, XP_(—)522245.1, BAB25732.1, NP_(—)001003854.1, AAC14163.1, CAI26289.1, CAG00953.1, YP_(—)227525.1, BAB13940.1, AAH84840.1, CAE51388.1, CAF25181.1, Q6 KB54, NP_(—)001002882.1, AAH23312.1, NP_(—)998924.1, CAH89922.1, AAF18019.1, NP_(—)001002883.1, AA013870.1, AA013869.1, AA013867.1, AA013866.1, AA013861.1, AA013859.1, NP_(—)777631.1, NP_(—)777628.1, and NP_(—)777623.1.

In one embodiment, said enzyme having sialyltransferase activity is obtained using the combinatorial DNA library approach of the invention as described herein. In one embodiment, said enzyme having sialyltransferase activity is a fusion protein. In a preferred embodiment, said fusion protein comprises a sialyltransferase catalytic domain and a cellular targeting signal peptide to target the sialyltransferase activity to the Golgi apparatus of the host cell. In one embodiment, said cellular targeting signal peptide is derived from the Mnn2 (leader 53) gene. More preferably, said cellular targeting signal peptide is encoded by the first 108 bases of Mnn2 (leader 53) from Genbank Accession Number NP_(—)009571.

Host cells may comprise a cellular pool of CMP-sialic acid that may be enhanced. In another embodiment, said host cell lacks an endogenous cellular pool of CMP-sialic acid, and may be modified to express CMP-sialic acid. In another embodiment, said host cell has been modified to express one or more enzyme activities involved in the CMP-Sia pathway. (See Example 16.)

In other embodiments, said host cell may be further modified to express one or more glycosylation enzymes selected from the group consisting of glycosyltransferases, glycosidases such as mannosidases, sugar transporters and the like. In yet other embodiments, said host cell may be modified to produce a glycoprotein comprising a terminal galactose. In other embodiments, said host cell may be modified to produce a glycoprotein comprising the Gal₍₁₋₄₎GlcNAc₍₁₋₄₎Man₍₃₋₅₎GlcNAc₂. In a preferred embodiment, said host has been modified to produce a complex glycoprotein comprising Gal₂GlcNAc₂Man₃GlcNAc₂ or a hybrid glycoprotein comprising Gal₁GlcNAc₁Man₅GlcNAc₂.

In other embodiments, the invention provides a method for producing a sialylated glycoprotein in a recombinant non-human eukaryotic host cell comprising introducing into the host a nucleic acid encoding an enzyme having sialyltransferase activity; and further comprising introducing into the host cell one or more additional nucleic acids encoding one or more enzymes involved in the biosynthesis or transport of CMP-Sialic acid.

In other embodiments, the host cell to be used in the methods described herein lacks the activity of one or more enzymes selected from the group consisting of mannosyltransferases and phosphomannosyltransferases. In other embodiments, the host cell to be used in the methods described herein is an OCH1 mutant of P. pastoris.

In other embodiments, the host cell to be used in the methods described herein is selected from the group consisting of: any Pichia sp., including but limited to: Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis and Pichia methanolica; any Saccharomyces sp. including but not limited to Saccharomyces cerevisiae Hansenula polymorpha; any Kluyveromyces sp. including but not limited to Kluyveromyces lactis; Candida albicans; any Aspergillus sp. species including but not limited to Aspergillus nidulans; Aspergillus niger; and Aspergillus oryzae; Trichoderma reseei; Chrysosporium lucknowense; any Fusarium sp. including but not limited to Fusarium gramineum and Fusarium venenatum; and Neurospora crassa.

The invention also provides a nucleic acid encoding a fusion protein comprising a sialyltransferase catalytic domain functionally linked to a cellular targeting signal. The invention also provides a host cell comprising said nucleic acid sequence. The invention further provides a non-human eukaryotic host cell comprising said nucleic acid, and further expressing CMP-sialic acid.

The invention further provides non-human host cell genetically engineered to produce a complex glycoprotein comprising NANA₍₁₋₄₎Gal₍₁₋₄₎GlcNAc₍₁₋₄₎Man₃GlcNAc₂ or a hybrid glycoprotein comprising NANAGalGlcNAcMan₅GlcNAc₂.

The invention also provides a recombinant glycoprotein produced by any one of the methods described herein.

Example 17 illustrates a method of the claimed invention, whereby a sialylated glycoprotein is produced in vivo.

Methods for In Vivo Transfer of Sialic Acid to N-Glycans Using Trans-Sialidase

An alternative method for obtaining sialylated proteins in non-human host cells that lack the CMP-Sia pathway is to use a trans-sialidase to transfer sialic acid onto an acceptable substrate glycoprotein. Protozoa, such as Trypanosoma cruzi, possess a cell-surface trans-sialidase which transfers sialic acid from host glycoproteins in an α 2,3 linkage to cell surface glycoproteins and glycolipids in a CMP-independent manner (see, e.g., Parodi, 1993, Schenkman, 1994). The gene encoding trans-sialidase contains an amino terminal region with catalytic activity and a C-terminal region with a plasma membrane anchoring domain (Pereira, 1991). T. cruzi trans-sialidase expressed in the baculovirus-insect cell system in the presence of sialic acid donors results in the sialylation of exogenous galactosylated acceptors (Marchal, I. 2001). T. cruzi trans-sialidase has also been expressed in P. pastoris (Laroy, W. 2000) as a means for obtaining high amounts of trans-sialidase for study. Thus, trans-sialidase can be used in host cells (e.g., a fungal host such as P. pastoris) engineered herein, to produce recombinant proteins having desirable N-glycan structures, as a means for sialylating glycoproteins with attached oligosaccharides bearing terminal galactose. Trans-sialidases other than that found in T. cruzi may be used. Transialidases suitable for use in the present invention are described for example in Colli, 1993. The disclosures of these references are hereby incorporated herein by reference.

Accordingly, the invention provides a method for producing a sialylated glycoprotein in a recombinant eukaryotic host cell comprising the step of introducing into the host a nucleic acid encoding an enzyme having trans-sialidase activity. The enzyme having trans-sialidase activity can be any enzyme having trans-sialidase activity including, but not limited to, T. cruzi trans-sialidase (e.g., GenBank Accession Nos. AJ276679, AJ002174), T. rangeli trans-sialidase (e.g., GenBank Accession No. L14943); T. brucei trans-sialidase (e.g., GenBank Accession No. XM_(—)340626), and T. carasasii trans-sialidase (e.g., GenBank Accession No. AY249142, AY14111), or homologues and variants thereof. In one embodiment, said method further comprises supplementing the medium for growing the host cell with a sialic acid donor.

In one embodiment, said enzyme having trans-sialidase activity is a fusion protein. In a preferred embodiment, said enzyme having trans-sialidase activity is a fusion protein comprising a trans-sialidase catalytic domain functionally linked to a cellular targeting signal peptide. In one embodiment said cellular targeting signal peptide is capable of targeting the trans-sialidase activity to the cell wall of the host cell.

In another embodiment, said host cell has been engineered or selected to produce a glycoprotein comprising a terminal galactose. In another embodiment, said host cell has been modified to produce a glycoprotein comprising the structure Gal₍₁₋₄₎GlcNAc₍₁₋₄₎Man₍₂₋₅₎GlcNAc₂. In preferred embodiments, said host cell produces a complex glycoprotein comprising Gal₂GlcNAc₂Man₃GlcNAc₂ or a hybrid glycoprotein comprising Gal₁GlcNAc₁Man₅GlcNAc₂.

The invention provides a method for producing a sialylated glycoprotein in a recombinant eukaryotic host cell comprising introducing into the host a nucleic acid encoding an enzyme having trans-sialidase activity; and further comprising introducing into the host cell one or more additional nucleic acids encoding one or more enzymes selected from the group consisting of glycosyltransferases, glycosidases such as mannosidases, sugar transporters, and enzymes involved in the biosynthesis or transport of CMP-Sialic acid.

In one embodiment, said host cell further comprises a sialic acid donor. In one embodiment, a sialic acid donor is added to the medium used to grow the host cell. In other embodiments, one or more precursors to CMP-Sialic acid may be added to the medium. Such CMP-Sia precursors include glucosamine [GlcN]; GlcNAc, UDP-GlcNAc and ManNAc. As described earlier, in other embodiments, said host cell has been adapted to comprise one or more sugar precursors, such as glucosamine or ManNAc, UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine, CMP-N-acetylneuraminic acid, or UDP-galactose.

The invention also provides a method for producing a sialylated glycoprotein in a recombinant non-human eukaryotic host cell comprising introducing into a medium used for growing the host cell an enzyme having a trans-sialidase activity and a sialic acid donor. The sialic acid donor may be CMP-Sia or the presence of sialic acid donor may be increased by the addition of a sugar nucleotide precursor such as CMP-Sia, glucosamine, GlcNAc, UDP-GlcNAc and ManNAc. In addition, the host cell may be adapted to comprise one or more sugar precursors, such as glucosamine or ManNAc, UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine, CMP-N-acetylneuraminic acid, or UDP-galactose.

In one embodiment, the host cell to be used in the methods described herein is selected from the group consisting of: Pichia sp., including but limited to: Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis and Pichia methanolica; any Saccharomyces sp including but not limited to Saccharomyces cerevisiae; Hansenula polymorpha; any Kluyveromyces sp. including but not limited to Kluyveromyces lactis; Candida albicans; any Aspergillus sp. including but not limited to Aspergillus nidulans, Aspergillus niger and Aspergillus oryzae; Trichoderma reseei; Chrysosporium lucknowense; any Fusarium sp. including but not limited to Fusarium gramineum and Fusarium venenatum; and Neurospora crassa.

In one embodiment, the host cell to be used in the methods described herein lacks the activity of one or more enzymes selected from the group consisting of mannosyltransferases and phosphomannosyltransferases. In one embodiment, the host cell to be used in the methods described herein is an OCH1 mutant of P. pastoris.

Introduction into a host cell of trans-sialidase activity may also be useful even if a CMP-Sia pathway is endogenous or has been engineered into the host cell. Trans-sialidase activity can increase the yield of secreted sialic acid terminated glycoproteins by transferring sialic acid from a donor protein onto recombinantly produced glycoproteins that did not sialylate within the secretory pathway or which had incomplete sialylation.

Whether a trans-sialidase activity is used alone, or in conjunction with an engineered CMP-Sia pathway and sialyl transferase activity, trans-sialidase may be (1) engineered to reside in the cell wall by fusion with the C-terminal domain of heat shock protein 150 (Mattila, 1996) or to another cell wall anchored protein (Bgl2, CRH1 and SCW19, (Weig, 2004)); (2) fed into the culture medium; or (3) immobilized in a column for use in a chromatography step during protein purification. When the trans-sialidase is fed into the culture medium for growing a host, the trans-sialidase is preferentially bound to a resin for simplified removal during protein purification. When the trans-sialidase is immobilized in a column, the supernatant of the host cell producing a recombinant glycoprotein is passed through the column in the presence of a sialic acid donor to effectuate the in vitro transfer of sialic acid onto the recombinantly produced glycoprotein.

As any strategy to engineer the formation of complex N-glycans into a host cell such as a lower eukaryote involves both the elimination as well as the addition of particular glycosyltransferase activities, a comprehensive scheme will attempt to coordinate both requirements. Genes that encode enzymes that are undesirable serve as potential integration sites for genes that are desirable. For example, 1,6 mannosyltransferase activity is a hallmark of glycosylation in many known lower eukaryotes. The gene encoding alpha-1,6 mannosyltransferase (OCH1) has been cloned from S. cerevisiae and mutations in the gene give rise to a viable phenotype with reduced mannosylation. The gene locus encoding alpha-1,6 mannosyltransferase activity therefore is a prime target for the integration of genes encoding glycosyltransferase activity. In a similar manner, one can choose a range of other chromosomal integration sites that, based on a gene disruption event in that locus, are expected to: (1) improve the cells ability to glycosylate in a more human-like fashion, (2) improve the cells ability to secrete proteins, (3) reduce proteolysis of foreign proteins and (4) improve other characteristics of the process that facilitate purification or the fermentation process itself.

Host Cells

Although the present invention is exemplified using a P. pastoris host organism, it is understood by those skilled in the art that other eukaryotic host cells, including plants, algae, insects and other species of yeast and fungal hosts, may be altered as described herein to produce human-like glycoproteins. The techniques described herein for identification and disruption of undesirable host cell glycosylation genes, e.g. OCH1, is understood to be applicable for these and/or other homologous or functionally related genes in other eukaryotic host cells such as other yeast and fungal strains. Preferably, robust protein production strains of fungal hosts that are capable of performing well in an industrial fermentation process are selected. These strains include, without limitation: any Pichia sp. including but limited to: Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis and Pichia methanolica; any Saccharomyces sp. including but not limited to Saccharomyces cerevisiae; Hansenula polymorpha; any Kluyveromyces sp. including but not limited to Kluyveromyces lactis; Candida albicans; any Aspergillus sp. including but not limited to Aspergillus nidulans, Aspergillus niger, and Aspergillus oryzae; Trichoderma reseei; Chlysosporium lucknowense; any Fusarium sp. including but not limited to Fusarium gramineum and Fusarium venenatum; and Neurospora crassa.

Another aspect of the present invention thus relates to a non-human eukaryotic host strain expressing glycoproteins comprising modified N-glycans that resemble those made by human-cells. Performing the methods of the invention in species other than yeast and fungal cells is thus contemplated and encompassed by this invention. It is contemplated that a combinatorial nucleic acid library of the present invention may be used to select constructs that modify the glycosylation pathway in any eukaryotic host cell system. For example, the combinatorial libraries of the invention may also be used in plants, algae and insects, and in other eukaryotic host cells, including mammalian and human cells, to localize proteins, including glycosylation enzymes or catalytic domains thereof, in a desired location along a host cell secretory pathway. Preferably, glycosylation enzymes or catalytic domains and the like are targeted to a subcellular location along the host cell secretory pathway where they are capable of functioning, and preferably, where they are designed or selected to function most efficiently.

As described in Examples 19 and 20, plant and insect cells may be engineered to alter the glycosylation of expressed proteins using the combinatorial library and methods of the invention. Furthermore, glycosylation in mammalian cells, including human cells, may also be modified using the combinatorial library and methods of the invention. It may be possible, for example, to optimize a particular enzymatic activity or to otherwise modify the relative proportions of various N-glycans made in a mammalian host cell using the combinatorial library and methods of the invention.

Examples of modifications to glycosylation which can be affected using a method according to this embodiment of the invention are: (1) engineering a eukaryotic host cell to trim mannose residues from Man₈GlcNAc₂ to yield a Man₅GlcNAc₂ N-glycan; (2) engineering eukaryotic host cell to add an N-acetylglucosamine (GlcNAc) residue to Man₅GlcNAc₂ by action of GlcNAc transferase I; (3) engineering a eukaryotic host cell to functionally express an enzyme such as an N-acetylglucosaminyl Transferase (GnTI, GnTII, GnTIII, GnTIV, GnTV, GnTVI), mannosidase II, fucosyltransferase (FT), galactosyl tranferase (GalT) or a sialyltransferase (ST); (4) an engineering or CMP-Sia pathway.

The invention also provides a host cell which lacks (or lacks an efficient) endogenous CMP-Sia biosynthetic pathway and which expresses a functional recombinant CMP-Sia biosynthetic pathway. Preferably, the host produces a cellular pool of CMP-Sia that may be used as a donor molecule in the presence of a sialyltransferase and a glycan acceptor (e.g., Gal₂GlcNAc₂Man₃GlcNAc₂) in a sialylation reaction. Using the methods of the invention, a variety of different hosts producing CMP-Sia may be generated.

By repeating the method, increasingly complex glycosylation pathways can be engineered into a target host, such as a lower eukaryotic microorganism. In one preferred embodiment, the host organism is transformed two or more times with DNA libraries including sequences encoding glycosylation activities. Selection of desired phenotypes may be performed after each round of transformation or alternatively after several transformations have occurred. Complex glycosylation pathways can be rapidly engineered in this manner.

Target Glycoproteins and Glycoprotein Compositions

The methods described herein are useful for producing glycoproteins, especially glycoproteins used therapeutically in humans, as well as compositions comprising such glycoproteins. Glycoproteins having specific glycoforms may be especially useful, for example, in the targeting of therapeutic proteins. Glycoprotein compositions of the present invention will comprise predominantly a specific glycoform. For example, mannose-6-phosphate has been shown to direct proteins to the lysosome, which may be essential for the proper function of several enzymes related to lysosomal storage disorders such as Gaucher's, Hunter's, Hurler's, Scheie's, Fabry's and Tay-Sachs disease, to mention just a few. Likewise, the addition of one or more sialic acid residues to a glycan side chain may increase the lifetime of a therapeutic glycoprotein in vivo after administration. Accordingly, host cells (e.g., lower eukaryotic or mammalian) may be genetically engineered to increase the extent of terminal sialic acid in glycoproteins expressed in the cells. Alternatively, sialic acid may be conjugated to the protein of interest in vitro prior to administration using a sialic acid transferase and an appropriate substrate. Changes in growth medium composition may be employed in addition to the expression of enzyme activities involved in human-like glycosylation to produce glycoproteins more closely resembling human forms (Weikert, 1999; Werner, 1998; Andersen, 1994; Yang, 2000). Specific glycan modifications to monoclonal antibodies (e.g. the addition of a bisecting GlcNAc) have been shown to improve antibody dependent cell cytotoxicity (Umana, 1999), which may be desirable for the production of antibodies or other therapeutic proteins.

Therapeutic proteins are typically administered by injection, orally, pulmonary, or other means. Examples of suitable target glycoproteins which may be produced according to the invention include, without limitation: erythropoietin, cytokines such as interferon-α, interferon-β, interferon-γ, interferon-ω, TNFα and granulocyte-CSF, coagulation factors such as factor VIII, factor IX, and human protein C, antithrombin III and thrombopoietin soluble IgE receptor α-chain, IgG, IgG fragments, IgM, interleukins such as IL-1 ra, urokinase, chymase, urea trypsin inhibitor, IGF-binding protein, epidermal growth factor, growth hormone-releasing factor, annexin V fusion protein, angiostatin, vascular endothelial growth factor-2, myeloid progenitor inhibitory factor-1, osteoprotegerin, α-1 antitrypsin, DNase II, α-proteins, AAT, rhTBP-1 (onercept, aka TNF Binding protein 1), TACI-Ig (transmembrane activator and calcium modulator and cyclophilin ligand interactor), FSH (follicle stimulating hormone), GM-CSF, GLP-1 w/ and w/o FC (glucagon like protein 1) IL-1 receptor agonist, sTNFr (enbrel, aka soluble TNF receptor Fc fusion) ATIII, rhThrombin, glucocerebrosidase and CTLA4-Ig (Cytotoxic T Lymphocyte associated Antigen 4-Ig)

The following are examples which illustrate the compositions and methods of this invention. These examples should not be construed as limiting: the examples are included for the purposes of illustration only. For example, while the examples illustrate embodiments of the compositions and methods of this invention relating to complex glycoproteins, one skilled in the art will recognize that the methods and compositions illustrated in the examples may be adapted to compositions and methods relating to hybrid glycoproteins, as described earlier herein.

Example 1 Cloning and Disruption of the OCH1 Gene in P. pastoris

Generation of an OCH1 Mutant of P. pastoris

A 1215 bp ORF of the P. pastoris OCH1 gene encoding a putative α-1,6 mannosyltransferase was amplified from P. pastoris genomic DNA (strain X-33, Invitrogen, Carlsbad, Calif.) using the oligonucleotides 5′-ATGGCGAAGGCAGATGGCAGT-3′ (SEQ ID NO:7) and 5′-TTAGTCCTTCCAACTTCCTTC-3′ (SEQ ID NO:8) which were designed based on the P. pastoris OCH1 sequence (Japanese Patent Application Publication No. 8-336387). Subsequently, 2685 bp upstream and 1175 bp downstream of the ORF of the OCH1 gene were amplified from a P. pastoris genomic DNA library (Boehm, 1999) using the internal oligonucleotides 5′-ACTGCCATCTGCCTTCGCCAT-3′ (SEQ ID NO:9) in the OCH1 gene, and 5′-GTAATACGACTCACTATAGGGC-3′ T7 (SEQ ID NO:10) and 5′-AATTAACCCTCACTAAAGGG-3′ T3 (SEQ ID NO:11) oligonucleotides in the backbone of the library bearing plasmid lambda ZAP II (Stratagene, La Jolla, Calif.). The resulting 5075 bp fragment was cloned into the pCR2.1-TOPO vector (Invitrogen, Carlsbad, Calif.) and designated pBK9.

After assembling a gene knockout construct that substituted the OCH1 reading frame with a HIS4 resistance gene, P. pastoris was transformed and colonies were screened for temperature sensitivity at 37° C. OCH1 mutants of S. cerevisiae are temperature sensitive and are slow growers at elevated temperatures. One can thus identify functional homologs of OCH1 in P. pastoris by complementing an OCH1 mutant of S. cerevisiae with a P. pastoris DNA or cDNA library. About 20 temperature sensitive strains were further subjected to a colony PCR screen to identify colonies with a deleted och1 gene. Several och1 deletions were obtained.

The linearized pBK9.1, which has 2.1 kb upstream sequence and 1.5 kb down stream sequence of OCH1 gene cassette carrying Pichia HIS4 gene, was transformed into P. pastoris BK1 [GS115 (his4 Invitrogen Corp., San Diego, Calif.) carrying the human IFN-β gene in the AOX1 locus] to knock out the wild-type OCH1 gene. The initial screening of transformants was performed using histidine drop-out medium followed by replica plating to select the temperature sensitive colonies. Twenty out of two hundred histidine-positive colonies showed a temperature sensitive phenotype at 37° C. To exclude random integration of pBK9.1 into the Pichia genome, the 20 temperature-sensitive isolates were subjected to colony PCR using primers specific to the upstream sequence of the integration site and to HIS4 ORF. Two out of twenty colonies were och1 defective and further analyzed using a Southern blot and a Western blot indicating the functional och1 disruption by the och1 knock-out construct. Genomic DNA were digested using two separate restriction enzymes BglII and ClaI to confirm the och1 knock-out and to confirm integration at the open reading frame. The Western Blot showed och1 mutants lacking a discrete band produced in the GS115 wild type at 46.2 kDa.

Example 2 Engineering of P. pastoris with α-1,2-Mannosidase to Produce Man₅GlcNAc₂-Containing IFN-β Precursors

An α-1,2-mannosidase is required for the trimming of Man₅GlcNAc₂ to yield Man₅GlcNAc₂, an essential intermediate for complex N-glycan formation. While the production of a Man₅GlcNAc₂ precursor is essential, it is not necessarily sufficient for the production of hybrid and complex glycans because the specific isomer of Man₅GlcNAc₂ may or may not be a substrate for GnTI. An och1 mutant of P. pastoris is engineered to express secreted human interferon-β under the control of an aox promoter. A DNA library is constructed by the in-frame ligation of the catalytic domain of human mannosidase IB (an α-1,2-mannosidase) with a sub-library including sequences encoding early Golgi and ER localization peptides. The DNA library is then transformed into the host organism, resulting in a genetically mixed population wherein individual transformants each express interferon-β as well as a synthetic mannosidase gene from the library. Individual transformant colonies are cultured and the production of interferon is induced by addition of methanol. Under these conditions, over 90% of the secreted protein is glycosylated interferon-β.

Supernatants are purified to remove salts and low-molecular weight contaminants by C₁₈ silica reversed-phase chromatography. Desired transformants expressing appropriately targeted, active α-1,2-mannosidase produce interferon-β including N-glycans of the structure Man₅GlcNAc₂, which has a reduced molecular mass compared to the interferon-β of the parent strain. The purified interferon-β is analyzed by MALDI-TOF mass spectroscopy and colonies expressing the desired form of interferon-β are identified.

Example 3 Generation of an och1 Mutant Strain Expressing an α-1,2-Mannosidase, GnTI and GnTII for Production of a Human-Like Glycoprotein

The 1215 bp open reading frame of the P. pastoris OCH1 gene as well as 2685 bp upstream and 1175 bp downstream was amplified by PCR (see also WO 02/00879), cloned into the pCR2.1-TOPO vector (Invitrogen) and designated pBK9. To create an och1 knockout strain containing multiple auxotrophic markers, 100 μg of pJN329, a plasmid containing an och1::URA3 mutant allele flanked with SfiI restriction sites was digested with SfiI and used to transform P. pastoris strain JC308 (Cereghino et al. Gene 263 (2001) 159-169) by electroporation. Following incubation on defined medium lacking uracil for 10 days at room temperature, 1000 colonies were picked and re-streaked. URA⁺ clones that were unable to grow at 37° C., but grew at room temperature, were subjected to colony PCR to test for the correct integration of the och1::URA3 mutant allele. One clone that exhibited the expected PCR pattern was designated YJN153. The Kringle 3 domain of human plasminogen (K3) was used as a model protein. A Neo^(R) marked plasmid containing the K3 gene was transformed into strain YJN153 and a resulting strain, expressing K3, was named BK64-1.

Plasmid pPB103, containing the Kluyveromyces lactic MNN2-2 gene which encodes a Golgi UDP-N-acetylglucosamine transporter was constructed by cloning a blunt BglII-HindIII fragment from vector pDL02 (Abeijon et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 93:5963-5968) into BglII and BamHI digested and blunt ended pBLADE-SX containing the P. pastoris ADE1 gene (Cereghino et al. (2001) Gene 263:159-169). This plasmid was linearized with EcoNI and transformed into strain BK64-1 by electroporation and one strain confirmed to contain the MNN2-2 by PCR analysis was named PBP1.

A library of mannosidase constructs was generated, comprising in-frame fusions of the leader domains of several type I or type II membrane proteins from S. cerevisiae and P. pastoris fused with the catalytic domains of several α-1,2-mannosidase genes from human, mouse, fly, worm and yeast sources (see, e.g., WO02/00879, incorporated herein by reference). This library was created in a P. pastoris HIS4 integration vector and screened by linearizing with SalI, transforming by electroporation into strain PBP1, and analyzing the glycans released from the K3 reporter protein. One active construct chosen was a chimera of the 988-1296 nucleotides (C-terminus) of the yeast SEC12 gene fused with a N-terminal deletion of the mouse α-1,2-mannosidase IA gene (FIG. 3), which was missing the 187 nucleotides. A P. pastoris strain expressing this construct was named PBP2.

A library of GnTI constructs was generated, comprising in-frame fusions of the same leader library with the catalytic domains of GnTI genes from human, worm, frog and fly sources (WO 02/00879). This library was created in a P. pastoris ARG4 integration vector and screened by linearizing with AatII, transforming by electroporation into strain PBP2, and analyzing the glycans released from K3. One active construct chosen was a chimera of the first 120 bp of the S. cerevisiae MNN9 gene fused to a deletion of the human GnTI gene, which was missing the first 154 bp. A P. pastoris strain expressing this construct was named PBP3.

A library of GnTII constructs was generated, which comprised in-frame fusions of the leader library with the catalytic domains of GnTII genes from human and rat sources (WO 02/00879). This library was created in a P. pastoris integration vector containing the NST^(R) gene conferring resistance to the drug nourseothricin. The library plasmids were linearized with EcoRI, transformed into strain RDP27 by electroporation, and the resulting strains were screened by analysis of the released glycans from purified K3.

Materials for the Following Reactions

MOPS, sodium cacodylate, manganese chloride, UDP-galactose and CMP-N-acetylneuraminic acid were from Sigma. Trifluoroacetic acid (TFA) was from Sigma/Aldrich, Saint Louis, Mo. Recombinant rat α2,6-sialyltransferase from Spodoptera frugiperda and β1,4-galactosyltransferase from bovine milk were from Calbiochem (San Diego, Calif.). Protein N-glycosidase F, mannosidases, and oligosaccharides were from Glyko (San Rafael, Calif.). DEAE ToyoPearl resin was from TosoHaas. Metal chelating “HisBind” resin was from Novagen (Madison, Wis.). 96-well lysate-clearing plates were from Promega (Madison, Wis.). Protein-binding 96-well plates were from Millipore (Bedford, Mass.). Salts and buffering agents were from Sigma (St. Louis, Mo.). MALDI matrices were from Aldrich (Milwaukee, Wis.).

Protein Purification

Kringle 3 was purified using a 96-well format on a Beckman BioMek 2000 sample-handling robot (Beckman/Coulter Ranch Cucamonga, Calif.). Kringle 3 was purified from expression media using a C-terminal hexa-histidine tag. The robotic purification is an adaptation of the protocol provided by Novagen for their HisBind resin. Briefly, a 150 uL (μL) settled volume of resin is poured into the wells of a 96-well lysate-binding plate, washed with 3 volumes of water and charged with 5 volumes of 50 mM NiSO4 and washed with 3 volumes of binding buffer (5 mM imidazole, 0.5M NaCl, 20 mM Tris-HCL pH7.9). The protein expression media is diluted 3:2, media/PBS (60 mM PO4, 16 mM KCl, 822 mM NaCl pH7.4) and loaded onto the columns. After draining, the columns are washed with 10 volumes of binding buffer and 6 volumes of wash buffer (30 mM imidazole, 0.5M NaCl, 20 mM Tris-HCl pH7.9) and the protein is eluted with 6 volumes of elution buffer (1M imidazole, 0.5M NaCl, 20 mM Tris-HCl pH7.9). The eluted glycoproteins are evaporated to dryness by lyophilyzation.

Release of N-Linked Glycans

The glycans are released and separated from the glycoproteins by a modification of a previously reported method (Papac, 1998). The wells of a 96-well MultiScreen IP (Immobilon-P membrane) plate (Millipore) are wetted with 100 uL of methanol, washed with 3×150 uL of water and 50 uL of RCM buffer (8M urea, 360 mM Tris, 3.2 mM EDTA pH8.6), draining with gentle vacuum after each addition. The dried protein samples are dissolved in 30 uL of RCM buffer and transferred to the wells containing 10 uL of RCM buffer. The wells are drained and washed twice with RCM buffer. The proteins are reduced by addition of 60 uL of 0.1M DTT in RCM buffer for 1 hr at 37° C. The wells are washed three times with 300 uL of water and carboxymethylated by addition of 60 uL of 0.1M iodoacetic acid for 30 min in the dark at room temperature. The wells are again washed three times with water and the membranes blocked by the addition of 100 uL of 1% PVP 360 in water for 1 hr at room temperature. The wells are drained and washed three times with 300 uL of water and deglycosylated by the addition of 30 uL of 10 mM NH₄HCO₃ pH 8.3 containing one milliunit of N-glycanase (Glyko). After 16 hours at 37° C., the solution containing the glycans was removed by centrifugation and evaporated to dryness.

Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry

Molecular weights of the glycans were determined using a Voyager DE PRO linear MALDI-TOF (Applied Biosciences) mass spectrometer using delayed extraction. The dried glycans from each well were dissolved in 15 uL of water and 0.5 uL spotted on stainless steel sample plates and mixed with 0.5 uL of S-DHB matrix (9 mg/mL of dihydroxybenzoic acid, 1 mg/mL of 5-methoxysalicilic acid in 1:1 water/acetonitrile 0.1% TFA) and allowed to dry.

Ions were generated by irradiation with a pulsed nitrogen laser (337 nm) with a 4 ns pulse time. The instrument was operated in the delayed extraction mode with a 125 ns delay and an accelerating voltage of 20 kV. The grid voltage was 93.00%, guide wire voltage was 0.10%, the internal pressure was less than 5×10-7 torr, and the low mass gate was 875 Da. Spectra were generated from the sum of 100-200 laser pulses and acquired with a 2 GHz digitizer. Man₅GlcNAc₂ oligosaccharide was used as an external molecular weight standard. All spectra were generated with the instrument in the positive ion mode. The estimated mass accuracy of the spectra was 0.5%.

Example 4 Engineering a Strain to Produce Galactosyltransferase Galactosyltransferase Reaction

Approximately 2 mg of protein (r-K3:hPg [PBP6-5]) was purified by nickel-affinity chromatography, extensively dialyzed against 0.1% TFA, and lyophilized to dryness. The protein was redissolved in 150 μL of 50 mM MOPS, 20 mM MnCl2, pH7.4. After addition of 32.5 μg (533 nmol) of UDP-galactose and 4 mU of β 1,4-galactosyltransferase, the sample was incubated at 37° C. for 18 hours. The samples were then dialyzed against 0.1% TFA for analysis by MALDI-TOF mass spectrometry.

The spectrum of the protein reacted with galactosyltransferase showed an increase in mass consistent with the addition of two galactose moieties when compared with the spectrum of a similar protein sample incubated without enzyme. Protein samples were next reduced, carboxymethylated and deglycosylated with PNGase F. The recovered N-glycans were analyzed by MALDI-TOF mass spectrometry. The mass of the predominant glycan from the galactosyltransferase reacted protein was greater than that of the control glycan by a mass consistent with the addition of two galactose moieties (325.4 Da).

Bobrowicz et al. (2004), which is incorporated by reference herein, discloses engineering a strain of P. pastoris capable of producing glycoproteins containing terminal galactose. This strain expressed a β1, 4GalT, a UDP galactose transporter and UDP-galactose-4-epimerase using the methods of the invention, as disclosed herein. See also the disclosure of Davidson, U.S. Ser. No. 11/108,088, filed on Apr. 15, 2005, the disclosure of which is hereby incorporated by reference herein.

Example 5 Engineering a Strain to Express Functional and Active Mannosidase II

To generate a human-like glycoform, a microorganism is engineered to express a mannosidase II enzyme which removes the two remaining terminal mannoses from the structure GlcNAcMan₅GlcNAc₂ (see FIG. 1B). A DNA library including sequences encoding cis and medial Golgi localization signals is fused in-frame to a library encoding mannosidase II catalytic domains. The host organism is a strain, e.g. a yeast, that is deficient in hypermannosylation (e.g. an och1 mutant) and provides N-glycans having the structure GlcNAcMan₅GlcNAc₂ in the Golgi and/or ER. After transformation, organisms having the desired glycosylation phenotype are selected. An in vitro assay is used in one method. The desired structure GlcNAcMan₃GlcNAc₂ (but not the undesired GlcNAcMan₅GlcNAc₂) is a substrate for the enzyme GlcNAc Transferase II (see FIG. 1B). Accordingly, single colonies may be assayed using this enzyme in vitro in the presence of the substrate, UDP-GlcNAc. The release of UDP is determined either by HPLC or an enzymatic assay for UDP. Alternatively, radioactively labeled UDP-GlcNAc or MALDI-TOF may be used. See Davidson et al., WO05/00584, the disclosure of which is hereby incorporated by reference.

The foregoing in vitro assays are conveniently performed on individual colonies using high-throughput screening equipment. Alternatively a lectin binding assay is used. In this case the reduced binding of lectins specific for terminal mannoses allows the selection of transformants having the desired phenotype. For example, Galantus nivalis lectin binds specifically to terminal α-1,3-mannose, the concentration of which is reduced in the presence of operatively expressed mannosidase II activity. In one suitable method, G. nivalis lectin attached to a solid agarose support (available from Sigma Chemical, St. Louis, Mo.) is used to deplete the transformed population of cells having high levels of terminal α-1,3-mannose.

Example 6 Engineering a Strain to Express Sialyltransferase

The enzymes α2,3-sialyltransferase and α2,6-sialyltransferase add terminal sialic acid to galactose residues in nascent human N-glycans, leading to mature glycoproteins (see “α 2,3 ST; α2,6 ST” in FIG. 1B). In human cells, the reactions occur in the trans Golgi or TGN. Accordingly, a DNA library is constructed by the in-frame fusion of sequences encoding sialyltransferase catalytic domains with sequences encoding trans Golgi or TGN localization signals (Malissard, 2000; Borsig, 1995). The host organism is a strain, e.g. a yeast, that is deficient in hypermannosylation (e.g., an och1 mutant), which provides N-glycans having terminal galactose residues in the late Golgi or TGN, and provides a sufficient concentration of CMP-sialic acid in the late Golgi or TGN. Following transformation, transformants having the desired phenotype are selected, e.g., using a fluorescent antibody specific for N-glycans having a terminal sialic acid. In addition, the strains are engineered to produce the CMP-NANA precursors as described in Example 16.

Example 17 further illustrates the engineering of a strain that expresses α2,6 ST and CMP-Sia and is able to produce glycoproteins comprising the NANA₂Gal₂GlcNAc₂Man₃GlcNAc₂ glycoform.

Sialyltransferase Reaction

After resuspending the (galactosyltransferase reacted) (Example 4) proteins in 10 μL of 50 mM sodium cacodylate buffer pH6.0, 300 μg (488 nmol) of CMP-N-acetylneuraminic acid (CMP-NANA) dissolved in 15 μL of the same buffer, and 5 μL (2 mU) of recombinant α-2,6 sialyltransferase were added. After incubation at 37° C. for 15 hours, an additional 200 μg of CMP-NANA and 1 mU of sialyltransferase were added. The protein samples were incubated for an additional 8 hours and then dialyzed and analyzed by MALDI-TOF-MS as above. The spectrum of the glycoprotein reacted with sialyltransferase showed an increase in mass when compared with that of the starting material (the protein after galactosyltransferase reaction). The N-glycans were released and analyzed as above. The increase in mass of the two ion-adducts of the predominant glycan was consistent with the addition of two sialic acid residues (580 and 583 Da).

Example 7 Engineering a Strain to Express UDP-GlcNAc Transporter

The cDNA of human Golgi UDP-GlcNAc transporter has been cloned by Ishida and coworkers. (Ishida, N., et al. 1999 J. Biochem. 126(1): 68-77). Guillen and coworkers have cloned the canine kidney Golgi UDP-GlcNAc transporter by phenotypic correction of a Kluyveromyces lactis mutant deficient in Golgi UDP-GlcNAc transport. (Guillen, E., et al. 1998). Thus a mammalian Golgi UDP-GlcNAc transporter gene has all of the necessary information for the protein to be expressed and targeted functionally to the Golgi apparatus of yeast. These or other cloned transporter genes may be engineered into a host organism to provide UDP-GlcNAc substrates for efficient GnT reactions in the Golgi and/or ER of the host. FIG. 10B demonstrates the effect of a strain expressing a K. lactis UDP-GlcNAc transporter. In comparison to FIG. 10A, which lacks a UDP-GlcNAc transporter, the effect of adding a UDP-GlcNAc transporter shows a dramatic increase in the production of GlcNAcMan₅GlcNAc₂.

Example 8 Engineering a Strain to Express GDP-Fucose Transporter

The rat liver Golgi membrane GDP-fucose transporter has been identified and purified by Puglielli, L. and C. B. Hirschberg 1999 J. Biol. Chem. 274(50):35596-35600. The corresponding gene can be identified using standard techniques, such as N-terminal sequencing and Southern blotting using a degenerate DNA probe. The intact gene is then expressed in a host microorganism that also expresses a fucosyltransferase.

Example 9 Engineering a Strain to Express UDP-Galactose Transporter

Human UDP-galactose (UDP-Gal) transporter has been cloned and shown to be active in S. cerevisiae. (Kainuma, M., et al. 1999 Glycobiology 9(2): 133-141). A second human UDP-galactose transporter (hUGT1) has been cloned and functionally expressed in Chinese Hamster Ovary Cells. Aoki, K., et al. 1999 J. Biochem. 126(5): 940-950. Likewise, Segawa and coworkers have cloned a UDP-galactose transporter from Schizosaccharomyces pombe (Segawa, H., et al. 1999 Febs Letters 451(3): 295-298). These or other sequences encoding UDP-galactose transporter activities may be introduced into a host cell directly or may be used as a component of a sub-library of the invention to engineer a strain having increased UDP-galactose transporter activity.

Example 10 Engineering a Strain to Express CMP-Sialic Acid Transporter

Human CMP-sialic acid transporter (hCST) has been cloned and expressed in Lec 8 CHO cells by Aoki and coworkers (1999). Molecular cloning of the hamster CMP-sialic acid transporter has also been achieved (Eckhardt, 1997). The functional expression of the murine CMP-sialic acid transporter was achieved in Saccharomyces cerevisiae by Berninsone, 1997. These or other sequences encoding CMP-sialic acid transporter activities may be introduced into a host cell directly or may be used as a component of a sub-library of the invention to engineer a strain having increased CMP-sialic acid transporter activity.

Example 11 Engineering of P. pastoris to Produce Man₅GlcNA₂ as the Predominant N-Glycan Structure Using a Combinatorial DNA Library

An och1 mutant of P. pastoris (see Examples 1 and 3) was engineered to express and secrete proteins such as the kringle 3 domain of human plasminogen (K3) under the control of the inducible AOXI promoter. The Kringle 3 domain of human plasminogen (K3) was used as a model protein. A DNA fragment encoding the K3 was amplified using Pfu turbo polymerase (Strategene, La Jolla, Calif.) and cloned into EcoRI and XbaI sites of pPICZαA (Invitrogen, Carlsbad, Calif.), resulting in a C-terminal 6-His tag. In order to improve the N-linked glycosylation efficiency of K3 (Hayes, 1975), Pro₄₆ was replaced with Ser₄₆ using site-directed mutagenesis. The resulting plasmid was designated pBK64. The correct sequence of the PCR construct was confirmed by DNA sequencing.

A combinatorial DNA library was constructed by the in-frame ligation of murine α-1,2-mannosidase IB (Genbank: 6678787) and IA (Genbank: 6754619) catalytic domains with a sub-library including sequences encoding Cop II vesicle, ER, and early Golgi localization peptides according to Table 6. The combined DNA library was used to generate individual fusion constructs, which were then transformed into the K3 expressing host organism, resulting in a genetically mixed population wherein individual transformants each express K3 as well as a localization signal/mannosidase fusion gene from the library. Individual transformants were cultured and the production of K3 was induced by transfer to a methanol containing medium. Under these conditions, after 24 hours of induction, over 90% of the protein in the medium was K3. The K3 reporter protein was purified from the supernatant to remove salts and low-molecular weight contaminants by Ni-affinity chromatography. Following affinity purification, the protein was desalted by size exclusion chromatography on a Sephadex G10 resin (Sigma, St. Louis, Mo.) and either directly subjected to MALDI-TOF analysis described below or the N-glycans were removed by PNGase digestion as described below (Release of N-glycans) and subjected to MALDI-TOF analysis (Miele, 1997).

Following this approach, a diverse set of transformants were obtained; some showed no modification of the N-glycans compared to the och1 knockout strain; and others showed a high degree of mannose trimming (FIGS. 5D, 5E). Desired transformants expressing appropriately targeted, active α-1,2-mannosidase produced K3 with N-glycans of the structure Man₅GlcNAc₂. This confers a reduced molecular mass to the glycoprotein compared to the K3 of the parent och1 deletion strain, a difference which was readily detected by MALDI-TOF mass spectrometry (FIG. 5). Table 7 indicates the relative Man₅GlcNAc₂ production levels.

TABLE 7 A Representative Combinatorial DNA Library Of Sequences/Catalytic Domains Localization Exhibiting Relative Levels Of Man₅GlcNAc₂ Production. Catalytic Targeting peptide sequences Domains MNS1(s) MNS1(m) MNS1(l) SEC12(s) SEC12(m) Mouse mannosidase FB4 FB5 FB6 FB7 FB8 1A Δ187 ++ + − ++ ++++ Mouse mannosidase GB4 GB5 GB6 GB7 GB8 1B Δ58 ++ + + ++ + Mouse mannosidase GC4 GC5 GC6 GC7 GC8 1B Δ99 − +++ + + + Mouse mannosidase GD4 GD5 GD6 GD7 GD8 1B Δ170 − − − + +

TABLE 8 Another Combinatorial DNA Library Of Localization Exhibiting Sequences/Catalytic Domains Relative Levels Of Man₅GlcNAc₂ Production. Catalytic Targeting peptide sequences Domains VAN1(s) VAN1(m) VAN1(l) MNN10(s) MNN10(m) MNN10(l) C. elegans BC18-5 BC19 BC20 BC27 BC28 BC29 mannosidase +++++ ++++ +++ +++++ +++++ +++ 1B Δ80 C. elegans BB18 BB19 BB20 BB18 BB19 BB20 mannosidase +++++ +++++ ++++ +++++ +++++ ++++ 1B Δ31

Targeting peptides were selected from MNS I (SwissProt P32906) in S. cerevisiae (long, medium and short) (see supra Nucleic Acid Libraries; Combinatorial DNA Library of Fusion Constructs) and SEC12 (SwissProt P11655) in S. cerevisiae (988-1140 nucleotides: short) and (988-1296: medium). Although majority of the targeting peptide sequences were N-terminal deletions, some targeting peptide sequences, such as SEC12 were C-terminal deletions. Catalytic domains used in this experiment were selected from mouse mannosidase 1A with a 187 amino acid N-terminal deletion; and mouse mannosidase 1B with a 58, 99 and 170 amino acid deletion. The number of (+)s, as used herein, indicates the relative levels of Man₅GlcNA₂ production. The notation (−) indicates no apparent production of Man₅GlcNA₂. The notation (+) indicates less than 10% production of Man₅GlcNA₂. The notation (++) indicates about 10-20% production of Man₅GlcNA₂. The notation with (+++) indicates about 20-40% production of Man₅GlcNA₂. The notation with (++++) indicates about 50% production of Man₅GlcNA₂. The notation with (+++++) indicates greater than 50% production of Man₅GlcNA₂.

Table 9 shows relative amount of Man₅GlcNAc₂ on secreted K3. Six hundred and eight (608) different strains of P. pastoris, Δoch1 were generated by transforming them with a single construct of a combinatorial genetic library that was generated by fusing nineteen (19) α-1,2 mannosidase catalytic domains to thirty-two (32) fungal ER, and cis-Golgi leaders.

TABLE 9 Amount of Man₅GlcNAc₂ on secreted K3 (% of total glycans) Number of constructs (%) N.D.*  19 (3.1)   0-10% 341 (56.1) 10-20%  50 (8.2)  20-40&  75 (12.3) 40-60%  72 (11.8) More than 60%   51 (8.4)^(†) Total 608 (100) *Several fusion constructs were not tested because the corresponding plasmids could not be propagated into E. coli prior to transformation into P. pastoris. ^(†)Clones with the highest degree of Man ⁵ GlcNAc ² trimming (30/51) were further analyzed for mannosidase activity in the supernant of the medium. The majority (28/30) displayed detectable mannosidase activity in the supernatant (e.g. FIG. 4B). Only two constructs displayed high Man₅GlcNAc₂ levels, while lacking mannosidase activity in the medium (e.g. FIG. 4C).

Table 7 shows two constructs pFB8 and pGC5, among others, displaying Man₅GlcNA₂. Table 8 shows a more preferred construct, pBC18-5, a S. cerevisiae VAN1 (s) targeting peptide sequence (from SwissProt 23642) ligated in-frame to a C. elegans mannosidase IB (Genbank AN: CAA98114) 80 amino acid N-terminal deletion (Saccharomyces Van1 (s)/C. elegans mannosidase IB Δ80). This fusion construct also produces a predominant Man₅GlcNA₂ structure, as shown in FIG. 5E. This construct was shown to produce greater than 50% Man₅GlcNA₂ (+++++).

Generation of a Combinatorial Localization/Mannosidase Library:

Generating a combinatorial DNA library of α-1,2-mannosidase catalytic domains fused to targeting peptides required the amplification of mannosidase domains with varying lengths of N-terminal deletions from a number of organisms. To approach this goal, the full length open reading frames (ORFs) of α-1,2-mannosidases were PCR amplified from either cDNA or genomic DNA obtained from the following sources: Homo sapiens, Mus musculus, Drosophila melanogaster, Caenorhabditis elegans, Aspergillus nidulans and Penicillium citrinum. In each case, DNA was incubated in the presence of oligonucleotide primers specific for the desired mannosidase sequence in addition to reagents required to perform the PCR reaction. For example, to amplify the ORF of the M. musculus α-1,2-mannosidase IA, the 5′-primer ATGCCCGTGGGGGGCCTGTTGCCGCTCTTCAGTAGC (SEQ ID NO:12) and the 3′-primer TCATTTCTCTTGCCATCAATTTCCTTCTTCTGTTCACGG (SEQ ID NO:13) were incubated in the presence of Pfu DNA polymerase (Stratagene, La Jolla, Calif.) and amplified under the conditions recommended by Stratagene using the cycling parameters: 94° C. for 1 min (1 cycle); 94° C. for 30 sec, 68° C. for 30 sec, 72° C. for 3 min (30 cycles). Following amplification the DNA sequence encoding the ORF was incubated at 72° C. for 5 min with 1U Taq DNA polymerase (Promega, Madison, Wis.) prior to ligation into pCR2.1-TOPO (Invitrogen, Carlsbad, Calif.) and transformed into TOP10 chemically competent E. coli, as recommended by Invitrogen. The cloned PCR product was confirmed by ABI sequencing using primers specific for the mannosidase ORF.

To generate the desired N-terminal truncations of each mannosidase, the complete ORF of each mannosidase was used as the template in a subsequent round of PCR reactions wherein the annealing position of the 5′-primer was specific to the 5′-terminus of the desired truncation and the 3′-primer remained specific for the original 3′-terminus of the ORF. To facilitate subcloning of the truncated mannosidase fragment into the yeast expression vector, pJN347 (FIG. 2C) AscI and PacI restriction sites were engineered onto each truncation product, at the 5′- and 3′-termini respectively. The number and position of the N-terminal truncations generated for each mannosidase ORF depended on the position of the transmembrane (TM) region in relation to the catalytic domain (CD). For instance, if the stem region located between the TM and CD was less than 150 bp, then only one truncation for that protein was generated. If, however, the stem region was longer than 150 bp then either one or two more truncations were generated depending on the length of the stem region.

An example of how truncations for the M. musculus mannosidase IA (Genbank: 6678787) were generated is described herein, with a similar approach being used for the other mannosidases. FIG. 3 illustrates the ORF of the M. musculus α-1,2-mannosidase IA with the predicted transmembrane and catalytic domains being highlighted in bold. Based on this structure, three 5′-primers were designed (annealing positions underlined in FIG. 3) to generate the Δ65-, Δ105- and Δ187-N-terminal deletions. Using the Δ65 N-terminal deletion as an example the 5′-primer used was 5′-GGCGCGCCGACTCCTCCAAGCTGCTCAGCGGGGTCCTGTTCCAC-3′(SEQ ID NO:14) (with the AscI restriction site highlighted in bold) in conjunction with the 3′-primer 5′-CCTTAATTAATCATTTCTCTTTGCCATCAATTTCCTTCTTCTGTTCACGG-3′ (SEQ ID NO:15) (with the PacI restriction site highlighted in bold). Both of these primers were used to amplify a 1561 bp fragment under the conditions outlined above for amplifying the full length M. musculus mannosidase 1A ORF. Furthermore, like the product obtained for the full length ORF, the truncated product was also incubated with Taq DNA polymerase, ligated into pCR2.1-TOPO (Invitrogen, Carlsbad, Calif.), transformed into TOP10 and ABI sequenced. After having amplified and confirmed the sequence of the truncated mannosidase fragment, the resulting plasmid, pCR2.1-Δ65 m MannIA, was digested with AscI and PacI in New England Biolabs buffer #4 (Beverly, Mass.) for 16 h at 37° C. In parallel, the pJN347 (FIG. 2C) was digested with the same enzymes and incubated as described above. Post-digestion, both the pJN347 (FIG. 2C) back-bone and the truncated catalytic domain were gel extracted and ligated using the Quick Ligation Kit (New England Biolabs, Beverly, Mass.), as recommended by the manufacturers, and transformed into chemically competent DH5α cells (Invitrogen, Carlsbad, Calif.). Colony PCR was used to confirm the generation of the pJN347-mouse Mannosidase IAΔ65 construct.

Having generated a library of truncated α-1,2-mannosidase catalytic domains in the yeast expression vector pJN347 (FIG. 2C) the remaining step in generating the targeting peptide/catalytic domain library was to clone in-frame the targeting peptide sequences (FIG. 2). Both the pJN347-mannosidase constructs (FIG. 2D) and the pCR2.1TOPO-targeting peptide constructs (FIG. 2B) such as were incubated overnight at 37° C. in New England Biolabs buffer #4 in the presence of the restriction enzymes NotI and AscI. Following digestion, both the pJN347-mannosidase back-bone and the targeting peptide regions were gel-extracted and ligated using the Quick Ligation Kit (New England Biolabs, Beverly, Mass.), as recommended by the manufacturers, and transformed into chemically competent DH5α cells (Invitrogen, Carlsbad, Calif.). Subsequently, the pJN347-targeting peptide/mannosidase constructs were ABI sequenced to confirm that the generated fusions were in-frame. The estimated size of the final targeting peptide/alpha-1,2-mannosidase library contains over 1300 constructs generated by the approach described above. FIG. 2 illustrates construction of the combinatorial DNA library.

Engineering a P. pastoris OCH1 Knock-Out Strain with Multiple Auxotrophic Markers.

The first step in plasmid construction involved creating a set of universal plasmids containing DNA regions of the KEX1 gene of P. pastoris (Boehm et al. Yeast 1999 May; 15(7):563-72) as space holders for the 5′ and 3′ regions of the genes to be knocked out. The plasmids also contained the S. cerevisiae Ura-blaster (Alani et al., Genetics 116, 541-545. 1987) as a space holder for the auxotrophic markers, and an expression cassette with a multiple cloning site for insertion of a foreign gene. A 0.9-kb fragment of the P. pastoris KEX1-5′ region was amplified by PCR using primers GGCGAGCTCGGCCTACCCGGCCAAGGCTGAGATCATTTGTCCAGCTTCA GA (SEQ ID NO:16) and GCCCACGTCGACGGATCCGTTTAAACATCGATTGGAGAGGCTGACACC GCTACTA (SEQ ID NO:17) and P. pastoris genomic DNA as a template and cloned into the SacI, SalI sites of pUC19 (New England Biolabs, Beverly, Mass.). The resulting plasmid was cut with BamHI and SalI, and a 0.8-kb fragment of the KEX1-3′ region that had been amplified using primers CGGGATCCACTAGTATTTAAATCATATGTGCGAGTGTACAACTCTTCCC ACATGG (SEQ ID NO:18) and GGACGCGTCGACGGCCTACCCGGCCGTACGAGGAATTTCTCGG ATGACTCTTTTC (SEQ ID NO:19) was cloned into the open sites creating pJN262. This plasmid was cut with BamHI and the 3.8-kb BamHI, BglII fragment of pNKY51 (Alani et al. 1987) was inserted in both possible orientations resulting in plasmids pJN263 (FIG. 4A) and pJN284 (FIG. 4B).

An expression cassette was created with NotI and PacI as cloning sites. The GAPDH promoter of P. pastoris was amplified using primers CGGGATCCCTCGAGAGATCTTTTTTGTAGAAATGTCTTGGTGCCT (SEQ ID NO:20) and GGACATGCATGCACTAGTGCGGCCGCCACGTGATAGTTGTTCA ATTGATTGAAATAGGGACAA (SEQ ID NO:21) and plasmid pGAPZ-A (Invitrogen) as template and cloned into the BamHI, SphI sites of pUC19 (New England Biolabs, Beverly, Mass.) (FIG. 4B). The resulting plasmid was cut with SpeI and SphI and the CYC1 transcriptional terminator region (“TT”) that had been amplified using primers CCTTGCTAGCTTAATTAACCGCGGCACGTCCGACGGCGGCCCA CGGGTCCCA (SEQ ID NO:22) and GGACATGCATGCGGATCCCTTAAGAGCCGGCAGCTTGCAAATT AAAGCCTTCGAGCGTCCC (SEQ ID NO:23) and plasmid pPICZ-A (Invitrogen) as a template was cloned into the open sites creating pJN261 (FIG. 4B).

A knockout plasmid for the P. pastoris OCH1 gene was created by digesting pJN263 with SalI and SpeI and a 2.9-kb DNA fragment of the OCH1-5′ region, which had been amplified using the primers GAACCACGTCGACGGCCATTGCGGCCAAAACCTTTTTTCCTATT CAAACACAAGGCATTGC (SEQ ID NO:24) and CTCCAATACTAGTCGAAGATTATCTTCTACGGTGCCTGGACTC (SEQ ID NO:25) and P. pastoris genomic DNA as a template, was cloned into the open sites (FIG. 4C). The resulting plasmid was cut with EcoRI and PmeI and a 1.0-kb DNA fragment of the OCH1-3′ region that had been generated using the primers TGGAAGGTTTAAACAAAGCTAGAGTAAAATAGATATAGCGAG ATTAGAGAATG (SEQ ID NO:26) and AAGAATTCGGCTGGAAGGCCTTGTACCTTGATGTAGTTCCCGTT TTCATC (SEQ ID NO:27) was inserted to generate pJN298 (FIG. 4C). To allow for the possibility to simultaneously use the plasmid to introduce a new gene, the BamHI expression cassette of pJN261 (FIG. 4B) was cloned into the unique BamHI site of pJN298 (FIG. 4C) to create pJN299 (FIG. 4E).

The P. pastoris Ura3-blaster cassette was constructed using a similar strategy as described in Lu. P., et al. 1998 (Cloning and disruption of the β-isopropylmalate dehydrogenase gene (Leu2) of Pichia stipidis with URA3 and recovery of the double auxotroph. Appl. Microbiol. Biotechnol. 49, 141-146.) A 2.0-kb PstI, SpeI fragment of P. pastoris URA3 was inserted into the PstI, XbaI sites of pUC19 (New England Biolabs, Beverly, Mass.) to create pJN306 (FIG. 4D). Then a 0.7-kb Sad, PvuII DNA fragment of the lacZ open reading frame was cloned into the SacI, SmaI sites to yield pJN308 (FIG. 4D). Following digestion of pJN308 (FIG. 4D) with PstI, and treatment with T4 DNA polymerase, the SacI-PvuII fragment from lacZ that had been blunt-ended with T4 DNA polymerase was inserted generating pJN315 (FIG. 4D). The lacZ/URA3 cassette was released by digestion with Sad and SphI, blunt ended with T4 DNA polymerase and cloned into the backbone of pJN299 that had been digested with PmeI and AflII and blunt ended with T4 DNA polymerase. The resulting plasmid was named pJN329 (FIG. 4E).

A HIS4 marked expression plasmid was created by cutting pJN261 (FIG. 4F) with EcoICRI (FIG. 4F). A 2.7 kb fragment of the Pichia pastoris HIS4 gene that had been amplified using the primers GCCCAAGCCGGCCTTAAGGGATCTCCTGATGACTGACTCACTGATAATA AAAATACGG (SEQ ID NO:28) and GGGCGCGTATTTAAATACTAGTGGATCTATCGAATCTAAATGTAAGTTA AAATCTCTAA (SEQ ID NO:29) cut with NgoMIV and SwaI and then blunt-ended using T4 DNA polymerase, was then ligated into the open site. This plasmid was named pJN337 (FIG. 4F). To construct a plasmid with a multiple cloning site suitable for fusion library construction, pJN337 was cut with NotI and PacI and the two oligonucleotides GGCCGCCTGCAGATTTAAATGAATTCGGCGCGCCTTAAT (SEQ ID NO:30) and TAAGGCGCGCCGAATTCATTTAAATCTGCAGGGC (SEQ ID NO:31) that had been annealed in vitro were ligated into the open sites, creating pJN347 (FIG. 4F).

To create an och1 knockout strain containing multiple auxotrophic markers, 100 μg of pJN329 was digested with SfiI and used to transform P. pastoris strain JC308 (Cereghino, 2001) by electroporation. Following transformation, the URA dropout plates were incubated at room temperature for 10 days. One thousand (1000) colonies were picked and restreaked. All 1000 clones were then streaked onto 2 sets of URA dropout plates. One set was incubated at room temperature, whereas the second set was incubated at 37° C. The clones that were unable to grow at 37° C., but grew at room temperature, were subjected to colony PCR to test for the correct OCHI knockout. One clone that showed the expected PCR signal (about 4.5 kb) was designated YJN153.

Example 12 Characterization of the Combinatorial DNA Library

Positive transformants screened by colony PCR confirming integration of the mannosidase construct into the P. pastoris genome were subsequently grown at room temperature in 50 ml BMGY buffered methanol-complex medium consisting of 1% yeast extract, 2% peptone, 100 mM potassium phosphate buffer, pH 6.0, 1.34% yeast nitrogen base, 4×10⁻⁵% biotin, and 1% glycerol as a growth medium) until OD_(600nm) 2-6 at which point they were washed with 10 ml BMMY (buffered methanol-complex medium consisting of 1% yeast extract, 2% peptone, 100 mM potassium phosphate buffer, pH 6.0, 1.34% yeast nitrogen base, 4×10⁻⁵% biotin, and 1.5% methanol as a growth medium) media prior to induction of the reporter protein for 24 hours at room temperature in 5 ml BMMY. Consequently, the reporter protein was isolated and analyzed by mass spectrophotometry and HPLC to characterize its glycan structure. Using the targeting peptides in Table 6, mannosidase catalytic domains localized to either the ER or the Golgi showed significant level of trimming of a glycan predominantly containing Man₈GlcNAc₂ to a glycan predominantly containing Man₅GlcNAc₂. This is evident when the glycan structure of the reporter glycoprotein is compared between that of P. pastoris och1 knock-out in FIGS. 5C, 6C and the same strain transformed with M. musculus mannosidase constructs as shown in FIGS. 5D, 5E, 6D-6F. FIGS. 5 and 6 show expression of constructs generated from the combinatorial DNA library which show significant mannosidase activity in P. pastoris. Expression of pGC5 (Saccharomyces MNS1 (m)/mouse mannosidase IB Δ99) (FIGS. 5D, 6E) produced a protein which has approximately 30% of all glycans trimmed to Man₅GlcNAc₂, while expression of pFB8 (Saccharomyces SEC12 (m)/mouse mannosidase IA Δ187) (FIG. 6F) produced approximately 50% Man₅GlcNAc₂ and expression of pBC18-5 (Saccharomyces VAN1 (s)/C. elegans mannosidase IB Δ80) (FIG. 5E) produced 70% Man₅GlcNAc₂.

Release of N-Glycans

The glycans were released and separated from the glycoproteins by a modification of a previously reported method (Papac et al. 1998 Glycobiology 8, 445-454). After the proteins were reduced and carboxymethylated and the membranes blocked, the wells were washed three time with water. The protein was deglycosylated by the addition of 30 μl of 10 mM NH4HCO3 pH 8.3 containing one milliunit of N-glycanase (Glyko, Novato, Calif.). After 16 hr at 37° C., the solution containing the glycans was removed by centrifugation and evaporated to dryness.

Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry

After the N-glycans were released by PNGase digestion, they were analyzed by Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry. Molecular weights of the glycans were determined using a Voyager DE PRO linear MALDI-TOF (Applied Biosciences) mass spectrometer using delayed extraction. The dried glycans from each well were dissolved in 15 μl of water and 0.5 μl was spotted on stainless steel sample plates and mixed with 0.5 μl of S-DHB matrix (9 mg/ml of dihydroxybenzoic acid, 1 mg/ml of 5-methoxysalicilic acid in 1:1 water/acetonitrile 0.1% TFA) and allowed to dry. Ions were generated by irradiation with a pulsed nitrogen laser (337 nm) with a 4 ns pulse time. The instrument was operated in the delayed extraction mode with a 125 ns delay and an accelerating voltage of 20 kV. The grid voltage was 93.00%, guide wire voltage was 0.1%, the internal pressure was less than 5×10-7 torr, and the low mass gate was 875 Da. Spectra were generated from the sum of 100-200 laser pulses and acquired with a 500 MHz digitizer. Man₅GlcNAc₂ oligosaccharide was used as an external molecular weight standard. All spectra were generated with the instrument in the positive ion mode.

Example 13 Trimming In Vivo by Alpha-1,2-Mannosidase

To ensure that the novel engineered strains of Example 11 in fact produced the desired Man₅GlcNAc₂ structure in vivo, cell supernatants were tested for mannosidase activity (see FIGS. 7-9). For each construct/host strain described below, HPLC was performed at 30° C. with a 4.0 mm×250 mm column of Altech (Avondale, Pa., USA) Econosil-NH₂ resin (5 μm) at a flow rate of 1.0 ml/min for 40 min. In FIGS. 7 and 8, degradation of the standard Man₉GlcNAc₂ [b] was shown to occur resulting in a peak which correlates to Man₈GlcNAc₂. In FIG. 7, the Man₉GlcNAc₂ [b] standard eluted at 24.61 min and Man₅GlcNAc₂ [a] eluted at 18.59 min. In FIG. 8, Man₉GlcNAc₂ eluted at 21.37 min and Man₅GlcNAc₂ at 15.67 min. In FIG. 9, the standard Man₈GlcNAc₂ [b] was shown to elute at 20.88 min.

P. pastoris cells comprising plasmid pFB8 (Saccharomyces SEC12 (m)/mouse mannosidase IA Δ187) were grown at 30° C. in BMGY to an OD600 of about 10. Cells were harvested by centrifugation and transferred to BMMY to induce the production of K3 (kringle 3 from human plasminogen) under control of an AOX1 promoter. After 24 hours of induction, cells were removed by centrifugation to yield an essentially clear supernatant. An aliquot of the supernatant was removed for mannosidase assays and the remainder was used for the recovery of secreted soluble K3. A single purification step using CM-sepharose chromatography and an elution gradient of 25 mM NaAc, pH5.0 to 25 mM NaAc, pH5.0, 1M NaCl, resulted in a 95% pure K3 eluting between 300-500 mM NaCl. N-glycan analysis of the K3 derived glycans is shown in FIG. 6F. The earlier removed aliquot of the supernatant was further tested for the presence of secreted mannosidase activity. A commercially available standard of 2-aminobenzamide-labeled N-linked-type oligomannose 9 (Man9-2-AB) (Glyko, Novato, Calif.) was added to: BMMY (FIG. 7A), the supernatant from the above aliquot (FIG. 7B), and BMMY containing 10 ng of 75 mU/mL of α-1,2-mannosidase from Trichoderma reesei (obtained from Contreras et al., WO 02/00856 A2) (FIG. 7C). After incubation for 24 hours at room temperature, samples were analyzed by amino silica HPLC to determine the extent of mannosidase trimming.

P. pastoris cells comprising plasmid pGC5 (Saccharomyces MNS1 (m)/mouse mannosidase IB Δ99) were similarly grown and assayed. Cells were grown at room temperature in BMGY to an OD600 of about 10. Cells were harvested by centrifugation and transferred to BMMY to induce the production of K3 under control of an AOX1 promoter. After 24 hours of induction, cells were removed by centrifugation to yield an essentially clear supernatant. An aliquot of the supernatant was removed for mannosidase assays and the remainder was used for the recovery of secreted soluble K3. A single purification step using CM-sepharose chromatography and an elution gradient of 25 mM NaAc, pH5.0 to 25 mM NaAc, pH5.0, 1M NaCl, resulted in a 95% pure K3 eluting between 300-500 mM NaCl. N-glycan analysis of the K3 derived glycans is shown in FIG. 5D. The earlier removed aliquot of the supernatant was further tested for the presence of secreted mannosidase activity as shown in FIG. 8B. A commercially available standard of Man9-2-AB (Glyko, Novato, Calif.) were added to: BMMY (FIG. 8A), supernatant from the above aliquot (FIG. 8B), and BMMY containing 10 ng of 75 mU/mL of α-1,2-mannosidase from Trichoderma reesei (obtained from Contreras et al., WO 02/00856 A2) (FIG. 8C). After incubation for 24 hours at room temperature, samples were analyzed by amino silica HPLC to determine the extent of mannosidase trimming.

Man9-2-AB was used as a substrate and it is evident that after 24 hours of incubation, mannosidase activity was virtually absent in the supernatant of the pFB8 (Saccharomyces SEC12 (m)/mouse mannosidase IA Δ187) strain digest (FIG. 7B) and pGC5 (Saccharomyces MNS1 (m)/mouse mannosidase IB Δ99) strain digest (FIG. 8B) whereas the positive control (purified α-1,2-mannosidase from T. reesei obtained from Contreras) leads to complete conversion of Man₉GlcNAc₂ to Man₅GlcNAc₂ under the same conditions, as shown in FIGS. 7C and 8C. This is conclusive data showing in vivo mannosidase trimming in P. pastoris pGC5 strain; and pFB8 strain, which is distinctly different from what has been reported to date (Contreras et al., WO 02/00856 A2).

FIG. 9 further substantiates localization and activity of the mannosidase enzyme. P. pastoris comprising pBC18-5 (Saccharomyces VAN1 (s)/C. elegans mannosidase IB Δ80) was grown at room temperature in BMGY to an OD600 of about 10. Cells were harvested by centrifugation and transferred to BMMY to induce the production of K3 under control of an AOX1 promoter. After 24 hours of induction, cells were removed by centrifugation to yield an essentially clear supernatant. An aliquot of the supernatant was removed for mannosidase assays and the remainder was used for the recovery of secreted soluble K3. A single purification step using CM-sepharose chromatography and an elution gradient 25 mM NaAc, pH5.0 to 25 mM NaAc, pH5.0, 1M NaCl, resulted in a 95% pure K3 eluting between 300-500 mM NaCl. N-glycan analysis of the K3 derived glycans is shown in FIG. 5E. The earlier removed aliquot of the supernatant was further tested for the presence of secreted mannosidase activity as shown in FIG. 9B. A commercially available standard of Man8-2-AB (Glyko, Novato, Calif.) was added to: BMMY (FIG. 9A), supernatant from the above aliquot pBC18-5 (Saccharomyces VAN1 (s)/C. elegans mannosidase IB Δ80) (FIG. 9B), and BMMY containing media from a different fusion construct pDD28-3 (Saccharomyces MNN10 (m) (from SwissProt 50108)/H. sapiens mannosidase IB Δ99) (FIG. 9C). After incubation for 24 hours at room temperature, samples were analyzed by amino silica HPLC to determine the extent of mannosidase trimming. FIG. 9B demonstrates intracellular mannosidase activity in comparison to a fusion construct pDD28-3 (Saccharomyces MNN10 (m) H. sapiens mannosidase IB Δ99) exhibiting a negative result (FIG. 9C).

Example 14 pH Optimum Assay of Engineered α-1,2-Mannosidase

P. pastoris cells comprising plasmid pBB27-2 (Saccharomyces MNN10 (s) (from SwissProt 50108)/C. elegans mannosidase IB Δ31) were grown at room temperature in BMGY to an OD600 of about 17. About 80 μL of these cells were inoculated into 600 μL BMGY and were grown overnight. Subsequently, cells were harvested by centrifugation and transferred to BMMY to induce the production of K3 (kringle 3 from human plasminogen) under control of an AOX1 promoter. After 24 hours of induction, cells were removed by centrifugation to yield an essentially clear supernatant (pH 6.43). The supernatant was removed for mannosidase pH optimum assays. Fluorescence-labeled Man₈GlcNAc₂ (0.5 μg) was added to 20 μL of supernatant adjusted to various pH (FIG. 11) and incubated for 8 hours at room temperature. Following incubation the sample was analyzed by HPLC using an Econosil NH₂ 4.6×250 mm, 5 micron bead, amino-bound silica column (Altech, Avondale, Pa.). The flow rate was 1.0 ml/min for 40 mM and the column was maintained to 30° C. After eluting isocratically (68% A:32% B) for 3 min, a linear solvent gradient (68% A:32% B to 40% A:60% B) was employed over 27 mM to elute the glycans (18). Solvent A (acetonitrile) and solvent B (ammonium formate, 50 mM, pH 4.5. The column was equilibrated with solvent (68% A:32% B) for 20 mM between runs.

Example 15 Engineering of P. pastoris to Produce N-Glycans with the Structure GlcNAcMan₅GlcNAc₂

GlcNAc Transferase I activity is required for the maturation of complex and hybrid N-glycans (U.S. Pat. No. 5,834,251). Man₅GlcNAc₂ may only be trimmed by mannosidase II, a necessary step in the formation of human glycoforms, after the addition of N-acetylglucosamine to the terminal α-1,3 mannose residue of the trimannose stem by GlcNAc Transferase I (Schachter, 1991 Glycobiology 1(5):453-461). Accordingly, a combinatorial DNA library was prepared including DNA fragments encoding suitably targeted catalytic domains of GlcNAc Transferase I genes from C. elegans and Homo sapiens; and localization sequences from GLS, MNS, SEC, MNN9, VAN1, ANP1, HOC1, MNN10, MNN11, MNT1, KTR1, KTR2, MNN2, MNN5, YUR1, MNN1, and MNN6 from S. cerevisiae and P. pastoris putative α-1,2-mannosyltransferases based on the homology from S. cerevisiae: D2, D9 and J3, which are KTR homologs. Table 10 includes but does not limit targeting peptide sequences such as SEC and OCH1, from P. pastoris and K. lactis GnTI, (See Table 6 and Table 10)

TABLE 10 A Representative Combinatorial Library Of Targeting Peptide Sequences/Catalytic Domain For UDP-N-Acetylglucosaminyl Transferase I (GnTI) Catalytic Targeting peptide Domain OCHI(s) OCHI(m) OCHI(l) MNN9(s) MNN9(m) Human, GnTI, Δ38 PB105 PB106 PB107 PB104 N/A Human, GnTI, Δ86 NB12 NB13 NB14 NB15 NB C. elegans, GnTI, Δ88 OA12 OA13 OA14 OA15 OA16 C. elegans, GnTI, Δ35 PA12 PA13 PA14 PA15 PA16 C. elegans, GnTI, Δ63 PB12 PB13 PB14 PB15 PB16 X. leavis, GnTI, Δ33 QA12 QA13 QA14 QA15 QA16 X. leavis, GnTI, Δ103 QB12 QB13 QB14 QB15 QB 16

Targeting peptide sequences were selected from OCHI in P. pastoris (long, medium and short) (see Example 11) and MNN9 (SwissProt P39107) in S. cerevisiae short, and medium. Catalytic domains were selected from human GnTI with a 38 and 86 amino acid N-terminal deletion, C. elegans (gly-12) GnTI with a 35 and 63 amino acid deletion as well as C. elegans (gly-14) GnTI with a 88 amino acid N-terminal deletion and X. leavis GnTI with a 33 and 103 amino acid N-terminal deletion, respectively.

A portion of the gene encoding human N-acetylglucosaminyl Transferase I (MGATI, GenBank Accession No. NM002406), lacking the first 154 bp, was amplified by PCR using oligonucleotides 5′-TGGCAGGCGCGCCTCAGTCAGCGCTCTCG-3′ (SEQ ID NO:32) and 5′-AGGTTAATTA AGTGCTAATTCCAGCTAGG-3′ (SEQ ID NO:33) and vector pHG4.5 (ATCC # 79003) as template. The resulting PCR product was cloned into pCR2.1-TOPO and the correct sequence was confirmed. Following digestion with AscI and PacI the truncated GnTI was inserted into plasmid pJN346 to create pNA. After digestion of pJN271 with NotI and AscI, the 120 bp insert was ligated into pNA to generate an in-frame fusion of the MNN9 transmembrane domain with the GnTI, creating pNA15.

The host organism is a strain of P. pastoris that is deficient in hypermannosylation (e.g. an och1 mutant), provides the substrate UDP-GlcNAc in the Golgi and/or ER (i.e. contains a functional UDP-GlcNAc transporter), and provides N-glycans of the structure Man₅GlcNAc₂ in the Golgi and/or ER (e.g. P. pastoris pFB8 (Saccharomyces SEC12 (m)/mouse mannosidase IA Δ187) from above). First, P. pastoris pFB8 was transformed with pPB103 containing the Kluyveromyces lactis MNN2-2 gene (Genbank AN: AF 106080) (encoding UDP-GlcNAc transporter) cloned into BamHI and BglII site of pBLADE-SX plasmid (Cereghino, 2001). Then the aforementioned combinatorial DNA library encoding a combination of exogenous or endogenous GnTI/localization genes was transformed and colonies were selected and analyzed for the presence of the GnTI construct by colony PCR. Our transformation and integration efficiency was generally above 80% and PCR screening can be omitted once robust transformation parameters have been established.

Protein Purification

K3 was purified from the medium by Ni-affinity chromatography utilizing a 96-well format on a Beckman BioMek 2000 laboratory robot. The robotic purification is an adaptation of the protocol provided by Novagen for their HisBind resin. Another screening method may be performed using a specific terminal GlcNAc binding antibody, or a lectin such as the GSII lectin from Griffonia simplificolia, which binds terminal GlcNAc (EY Laboratories, San Mateo, Calif.). These screens can be automated by using lectins or antibodies that have been modified with fluorescent labels such as FITC or analyzed by MALDI-TOF.

Secreted K3 can be purified by Ni-affinity chromatography, quantified and equal amounts of protein can be bound to a high protein binding 96-well plate. After blocking with BSA, plates can be probed with a GSII-FACS lectin and screened for maximum fluorescent response. A preferred method of detecting the above glycosylated proteins involves the screening by MALDI-TOF mass spectrometry following the affinity purification of secreted K3 from the supernatant of 96-well cultured transformants. Transformed colonies were picked and grown to an OD600 of 10 in a 2 ml, 96-well plate in BMGY at 30° C. Cells were harvested by centrifugation, washed in BMMY and resuspended in 250 ul of BMMY. Following 24 hours of induction, cells were removed by centrifugation, the supernatant was recovered and K3 was purified from the supernatant by Ni affinity chromatography. The N-glycans were released and analyzed by MALDI-TOF delayed extraction mass spectrometry as described herein.

In summary, the methods of the invention yield strains of P. pastoris that produce GlcNAcMan₅GlcNAc₂ in high yield, as shown in FIG. 10B. At least 60% of the N-glycans are GlcNAcMan₅GlcNAc₂. To date, no report exists that describes the formation of GlcNAcMan₅GlcNAc₂ on secreted soluble glycoproteins in any yeast. Results presented herein show that addition of the UDP-GlcNAc transporter along with GnTI activity produces a predominant GlcNAcMan₅GlcNAc₂ structure, which is confirmed by the peak at 1457 (m/z) (FIG. 10B).

Construction of Strain PBP-3:

The P. pastoris strain expressing K3, (Δoch1, arg-, ade-, his-) was transformed successively with the following vectors. First, pFB8 (Saccharomyces SEC12 (m)/mouse mannosidase IA Δ187) was transformed in the P. pastoris strain by electroporation. Second, pPB103 containing Kluyveromyces lactis MNN2-2 gene (Genbank AN: AF106080) (encoding UDP-GlcNAc transporter) cloned into pBLADE-SX plasmid (Cereghino et al. Gene 263 (2001) 159-169) digested with BamHI and BglII enzymes was transformed in the P. pastoris strain. Third, pPB104 containing Saccharomyces MNN9 (s)/human GnTI Δ38 encoding gene cloned as NotI-PacI fragment into pJN336 was transformed into the P. pastoris strain.

Example 16 Engineering a CMP-Sialic Acid Biosynthetic Pathway in P. pastoris Cloning Enzymes Involved in CMP-Sialic Acid Synthesis

One method for cloning a CMP-sialic acid biosynthetic pathway into a fungal host cell involves amplifying the E. coli NeuA, NeuB and NeuC genes from E. coli genomic DNA using the polymerase chain reaction in conjunction with primer pairs specific for each open reading frame (ORF) (Table 12 below, and FIGS. 17, 16 and 15, respectively).

For cloning a mammalian CMP-sialic acid biosynthetic pathway, the mouse CMP-Sia synthase ORF (FIG. 18) was amplified from a mouse pituitary cDNA library in conjunction with the primer pairs set forth in Table 12. The GlcNAc epimerase (previously discussed in an alternate method for producing CMP-Sia intermediates), was amplified from porcine cDNA using PCR in conjunction with primer pairs specific for the corresponding gene (Table 12 and FIG. 20). The sialate aldolase gene (FIG. 22) was amplified from E. coli genomic DNA using the polymerase chain reaction in conjunction with the primer pairs set forth in Table 12. The mouse bifunctional UDP-N-acetylglucosamine-2-Epimerase/N-acetylmannosamine kinase gene was amplified from mouse liver using the polymerase chain reaction in conjunction with the primer pairs set forth in Table 12. The mouse N-acetylneuraminate-9-phosphate synthase gene was amplified from mouse liver using the polymerase chain reaction in conjunction with the primer pairs set forth in Table 12. The human CMP-Sia synthase gene was amplified from human liver using the polymerase chain reaction in conjunction with the primer pairs set forth in Table 12. In each case, the ORFs were amplified using a high-fidelity DNA polymerase enzyme under the following thermal cycling conditions: 97° C. for 1 min, 1 cycle; 97° C. for 20 sec, 60° C. for 30 sec, 72° C. for 2 min, 25 cycles; 72° C. for 2 min, 1 cycle. Following DNA sequencing to confirm the absence of mutations, each ORF is re-amplified using primers containing compatible restriction sites to facilitate the subcloning of each into suitable fungal expression vectors.

TABLE 12 Primer name Primer sequence NeuA sense 5′-ATGAGAACAAAAATTATTGCGATAATTCCAGCCC G-3′ (SEQ ID NO: 45) NeuA antisense 5′-TCATTTAACAATCTCCGCTATTTCGTTTTC-3′ (SEQ ID NO: 46) NeuB sense 5′-ATGAGTAATATATATATCGTTGCTGAAATTGGTTG- 3′ (SEQ ID NO: 47) NeuB antisense 5′-TTATTCCCCCTGATTTTTGAATTCGCTATG-3′ (SEQ ID NO: 48) NeuC sense 5′-ATGAAAAAAATATTATACGTAACTGGATCTAGAG-3′ (SEQ ID NO: 49) NeuC antisense 5′- CTAGTCATAACTGGTGGTACATTCCGGGATGTC-3′ (SEQ ID NO: 50) mouse CMP-Sia 5′-ATGGACGCGCTGGAGAAGGGGGCCGTCACGTC- synthase sense 3′ (SEQ ID NO: 51) mouse CMP-Sia 5′- synthase antisense CTATTTTTGGCATGAGTTATTAACTTTTTCTATCA G-3′ (SEQ ID NO: 52) porcine GlcNAc 5′-ATGGAGAAGGAGCGCGAAACTCTGCAGG-3′ epimerase sense (SEQ ID NO: 53) porcine GlcNAc 5′-CTAGGCGAGGCGGCTCAGCAGGGCGCTC-3′ epimerase (SEQ ID NO: 54) antisense E. coli Sialate 5′-ATGGCAACGAATTTACGTGGCGTAATGGCTG-3′ aldolase sense (SEQ ID NO: 55) E. coli Sialate 5′-TCACCCGCGCTCTTGCATCAACTGCTGGGC-3′ aldolase antisense (SEQ ID NO: 56) mouse bifunctional 5′- UDP-N- ATGGAGAAGAACGGGAACAACCGAAAGCTCCG-3′ acetylglucosamine- (SEQ ID NO: 69) 2-epimerase/N- acetylmannosamine kinase sense mouse bifunctional 5′-CTAGTGGATCCTGCGCGTTGTGTAGTCCAG-3′ UDP-N- (SEQ ID NO: 70) acetylglucosamine- 2-epimerase/ N-acetylmannosamine kinase antisense mouse Sia9P syn 5′-ATGCCGCTGGAACTGGAGCTGTGTCCCGGGC-3′ sense (SEQ ID NO: 71) mouse Sia9P syn 5′-TTAAGCCTTGATTTTCTTGCTGTGACTTTCCAC- antisense 3′ (SEQ ID NO: 72) human N- 5′- acetylneuraminic GGGAGAATGCGGCCGCCACCATGGGGCTGAGCCG acid phosphatase C GTGCGGGCGGTTTTC-3′ (SEQ ID NO: 73) (NANP) sense human N- 5′ - acetylneuraminic GTATAGACTGCAAAGTCAGTATGTCCACTTGATT acid phosphatase AATTAACC-3′ (SEQ ID NO: 74) (NANP) antisense human CMP-Sia 5′-ATGGACTCGGTGGAGAAGGGGGCCGCCACC-3′ synthase sense (SEQ ID NO: 75) human CMP-Sia 5′-CTATTTTTGGCATGAATTATTAACTTTTTCC-3′ synthase antisense (SEQ ID NO: 76) Expression of Bacterial Neu Genes in P. pastoris

The 1176 bp PCR amplified fragment of the NeuC gene was ligated into the NotI-AscI site in the yeast integration vector pJN348 (a modified pUC19 vector comprising a GAPDH promoter, a NotI AscI PacI restriction site cassette, CycII transcriptional terminator, URA3 as a positive selection marker) producing pSH256. Similarly, the PCR amplified fragment (1041 bp) of the NeuB gene was ligated into the NotI-PacI site in the yeast integration vector pJN335, under the control of a GAPDH promoter using ADE as a positive selection marker, producing pSH255. The 1260 bp PCR amplified fragment of the NeuA gene was ligated into the NotI-PacI site in the yeast integration vector pJN346, under the control of a GAPDH promoter with ARG as a positive selection marker, to produce pSH254. After transforming P. pastoris with each vector by electroporation, the cells were plated onto the corresponding drop-out agar plates to facilitate positive selection of the newly introduced vector(s). To confirm the introduction of each gene, several hundred clones were repatched onto the respective dropout plates and grown for two days at 26° C. Once sufficient material had grown, each clone was screened by colony PCR using primers specific for the introduced gene. Conditions for colony PCR using the polymerase ExTaq™ from Takara (Madison, Wis.), were as follows: 97° C. for 3 min, 1 cycle; 97° C. for 20 sec, 50° C. for 30 sec, 72° C. for 2 min/kb, 30 cycles; 72° C. for 10 min, 1 cycle. Subsequently, several positive clones from colony PCR were grown in a baffled flask containing 200 ml of growth media. The base composition of growth media containing 2.68 g/l yeast nitrogen base, 200 mg/l biotin and 2 g/l dextrose was supplemented with appropriate amino acids depending on the strain used. The cells were grown in this media in the presence or absence of 20 mM ManNAc. Following growth in the baffle flask at 30° C. for 4-6 days, the cells were pelleted and analyzed for intermediates of the sialic acid pathway, as described below in this Example.

Expression of GlcNAc Epimerase Gene in P. pastoris

The PCR amplified fragment of the porcine GlcNAc epimerase gene is ligated into the NotI-PacI site in the yeast integration vector pJN348, under the control of the GAPDH promoter using URA3 as a positive selection marker. The P. pastoris strain producing endogenous GlcNAc is transformed with the vector carrying the GlcNAc epimerase gene fragment and screened for transformants.

Expression of Sialate Aldolase Gene in P. pastoris

The PCR amplified fragment of the E. coli sialate aldolase gene is ligated into the NotI-PacI site in the yeast integration vector pJN335, under the control of the GAPDH promoter with ADE as a positive selection marker, producing pSH275. The P. pastoris strain producing ManNAc is transformed with the vector carrying the sialate aldolase gene fragment and screened for transformants.

Expression of the Gene Encoding UDP-N-Acetylglucosamine-2-Epimerase/N-Acetylmannosamine Kinase in P. pastoris

The PCR amplified fragment of the gene encoding the mouse bifunctional UDP-N-acetylglucosamine-2-Epimerase/N-acetylmannosamine Kinase enzyme was ligated into the NotI-PacI site in the yeast integration vector pJN348, under the control of the GAPDH promoter using URA as a positive selection marker, producing pSH284. The P. pastoris strain producing ManNAc was transformed with the vector carrying the gene fragment and screened for transformants.

Expression of the Gene Encoding N-Acetyl-Neuraminate-9-Phosphate Synthase in P. pastoris

The PCR amplified fragment of the mouse N-acetylneuraminate-9-phosphate synthase gene was ligated into the NotI-PacI site in the yeast integration vector pJN335, under the control of the GAPDH promoter with ADE as a positive selection marker producing, pSH285. The P. pastoris strain producing ManNAc-6-P was transformed with the vector carrying the above gene fragment and screened for transformants.

Identification, Cloning and Expression of the Gene Encoding N-Acetylneuraminate-9-Phosphatase

N-acetylneuraminate-9-phosphatase activity has been detected in the cytosolic fraction of rat liver cells (Van Rinsum, 1984). We have repeated this method and isolated a cell extract fraction containing phosphatase activity selective against NeuAc-9-P. SDS-PAGE electrophoresis of this fraction identifies a single protein band. Subsequently, this sample was electroblotted onto a PDVF membrane, and the N-terminal amino acid sequence was identified by Edman degradation. The sequence identified allows the generation of degenerate oligonucleotides for the 5′-terminus of the ORF of the isolated protein. Using these degenerate primers in conjunction with the AP1 primer supplied in a rat liver Marathon-ready cDNA library (Clontech) according to the manufacturer's instructions, a full length ORF was isolated. The complete ORF was subsequently ligated into the yeast integration vector pJN347, under the control of the GAPDH promoter using a HIS gene as a positive selection marker. The P. pastoris strain producing NeuAc-9-P was transformed with the vector carrying the desired gene fragment and screened for transformants as described above in this Example.

Cloning and Expression of a CMP-Sialic Acid Synthase Gene in P. pastoris

The PCR amplified fragment of the mouse CMP-Sia synthase gene was ligated into the NotI-PacI site in the yeast integration vector pJN346 under the control of the GAPDH promoter with the ARG gene as a positive selection marker. A P. pastoris strain producing sialic acid was transformed with the vector carrying the above gene fragment and screened for transformants as described above in this Example. Likewise, the human CMP-Sia synthase gene (Genbank AN AF397212) was amplified and ligated into the NotI-PacI site of the yeast expression vector pJN346 under the control of a GAPDH promoter with ARG as a positive selection marker, producing the vector pSH257. A P. pastoris strain capable of producing sialic acid is transformed with pSH257 by electroporation, producing a strain capable of generating CMP-Sia.

Expression of the Hybrid CMP-Sia Pathway in P. pastoris

The P. pastoris strain JC308 (Cereghino, 2001) was super-transformed or simultaneously transformed with 20 mg of each of the vectors containing NeuC (pSH256), NeuB (pSH255) and hCMP-Sia synthase (pSH257) by electroporation. The resulting cells were plated on minimal media supplemented with histidine (containing 1.34 g/l yeast nitrogen base, 200 mg/l biotin, 2 g/l dextrose, 20 g/l agar and 20 mg/l L-histidine). Following incubation at 30° C. for 4 days, several hundred clones were isolated by repatching onto minimal media plates supplemented with histidine (see above for composition). The repatched clones were grown for 2 days prior to performing colony PCR (as described above in this Example) on the clones. Primers specific for NeuC, NeuB and hCMP-Sia synthase were used to confirm the presence of each ORF in the transformed clones. Twelve clones positive for all three ORFS (designated YSH99a-1) were grown in a baffled flask containing 200 ml of growth media (containing 2.68 g/l yeast nitrogen base, 200 mg/l biotin, 20 mg/l L-histidine and 2 g/l dextrose). The effect of supplementing the growth media with ManNAc was investigated by growing the cells in the presence or absence of 20 mM ManNAc. Following growth in the baffle flask at 30° C. for 4-6 days the cells are pelleted and analyzed for the presence of sialic acid pathway intermediates as described below in this Example.

Comparing the cell extracts using the assay outlined below in this Example, the cell extracts from P. pastoris YSH99a grown without exogenous CMP-Sia, showed transfer of Sia onto acceptor substrates, indicating the presence of endogenous CMP-Sia production (FIG. 25). Both mono- and di-sialylated biantennary N-glycans eluted at 20 min and 23 mM, their respective corresponding time. Additionally, subsequent sialidase treatment showed the removal of sialic acid (FIG. 26). Thus, a yeast strain engineered with a hybrid CMP-Sia biosynthetic pathway as described above, containing the NeuC, NeuB and hCMP-Sia synthase, was shown to be capable of generating an endogenous pool of CMP-sialic acid.

Assay for the Presence of Cytidine-5′-Monophospho-N-Acetylneuraminic Acid in Genetically Altered P. pastoris

Yeast cells were washed three times with cold PBS buffer, and suspended in 100 mM ammonium bicarbonate pH 8.5 and kept on ice. The cells were lysed using a French pressure cell followed by sonication. Soluble cell contents were separated from cell debris by ultracentrifugation. Ice cold ethanol was added to the supernatant to a final concentration of 60% and kept on ice for 15 minutes prior to removal of insoluble proteins by ultracentrifugation. The supernatant was frozen and concentrated by lyophilization. The dried sample was resuspended in water (ensuring pH is 8.0) and then filtered through a pre-rinsed 10,000 MWCO Centricon cartridge. The filtrate was separated on a Mono Q ion-exchange column according to manufacturer's instructions and the elution fractions that co-eluted with authentic CMP-sialic acid were pooled and lyophilized.

The dried filtrate was dissolved in 100 μL of 100 mM ammonium acetate pH6.5, 11 μL (5 mU) of α-2,6 sialyltransferase and 3.3 μL (12 mU) of α-2,3 sialyltransferase were added, and 10 μL of the mixture was removed for a negative control. Subsequently, 7 μL (1.4 μg) of 2-aminobenzamide-labeled asialo-biantennary N-glycan (NA2, Glyco Inc., San Rafael, Calif.) was added to the remaining mixture, followed by the removal of 10 μL for a positive control. The sample and control reactions were then incubated at 37° C. for 16 hr. 10 μL of each sample were then separated on a GlycoSep-C anion exchange column according to manufacturer's instructions. A separate control consisting of approximately 0.05 μg each of monosialylated and disialylated biantennary glycans was separated on the column to establish relative retention times. The results are shown in FIGS. 23-27.

Sialidase Treatment

The incubation of bi-antennary galactosylated N-glycans with an extract from the P. pastoris YSH99a strain in the presence of sialyltransferases produced sialylated N-glycans, which were subsequently desialylated as follows: a sialylated sample was passed through a Microcon cartridge, with 10,000 molecular weight cut-off, to remove the transferases. The cartridge was washed twice with 100 μl of water, which was pooled with the original eluate. Analysis of the eluate by HPLC (FIG. 26) produced a spectrum similar to the HPLC spectrum prior to the Microcon treatment. The remaining sample was lyophilized to dryness and resuspended in 25 μl of 1×NEB G1 buffer. After addition of 100 U of sialidase (New England Biolabs #P0720L, Beverley, Mass.), the resuspended sample was incubated overnight at 37° C. prior to HPLC analysis, as described previously.

Example 17 Engineering a P. pastoris Strain Capable of Transferring Sialic Acid to N-Glycan

A P. pastoris strain capable of transferring sialic acid to N-glycans in vivo has been created as described in detail herein.

Generation of pSH321b for Expressing Human UDP-GlcNAc-2-Epimerase/N-Acetylmannosamine Kinase (hGNE) and N-Acetylneuraminate-9-Phosphate Synthase (HsiaPsyn)

The gene sequence encoding the human UDP-GlcNAc-2-epimerase/N-acetylmannosamine kinase (hGNE; Genbank Accession No. AJ238764) was amplified as NotI-PacI fragment from human liver cDNA using oligonucleotide primers GGGAGAATGCGGCCGCCACCATGGAGAAGAATGGAAATAACCGAAAG CTGCG (hGNE NotI/Koz) (SEQ ID NO: 77) and CCTTAATTAACTAGTAGATCCTGCGTGTTGTGTAGTCCAGAAC (hGNE PacI rev) (SEQ ID NO: 78) with Advantage™ DNA polymerase (BD Biosciences) according to manufacturer's instructions. The conditions used for thermocycling were as follows: 95° C. 2 min, 1 cycle; 97° C. 30 sec, 60° C. 30 sec, 72° C. 5 min, 25 cycles; 72° C. 5 min. The resulting 2.2 Kb fragment was cloned into pCR2.1 (Invitrogen), sequenced and designated pSH281. The hGNE was cloned from pSH281 into pJN348 (as described in US Publication No. 2004/0230042) as a NotI-PacI fragment, producing vector pSH284. Positive clones from colony PCR were grown in a baffled flask containing 200 mL of BMGY media consisting of 2.68 g/L yeast nitrogen base, 200 mg/L biotin and 2 g/L dextrose, supplemented with amino acids depending on the strains.

The gene sequence encoding the human N-acetylneuraminate-9-phosphate synthase (hSiaPsyn; Genbank Accession No. AF257466) was amplified as above using primers GGGAGAATGCGGCCGCCACCATGCCGCTGGAGCTGGAGCTGTGTCCCG (hSiaPsyn NotI/Koz) (SEQ ID NO: 79) and CCTTAATTAATTAAGACTTGATTTTTTTGCCATGATTATCTACC (hSiaPsyn PacI rev) (SEQ ID NO: 80). The conditions used for thermocycling were 2.5 min extension instead of 5 min. The resulting 1.1 Kb fragment was cloned into pCR2.1 (Invitrogen), sequenced and designated pSH282. The hSiaPsyn was cloned from pSH282 into pJN664 (as disclosed in US Publication No. 2004/230042) as a NotI-PacI fragment, giving the vector pSH302. This construct was used as template to amplify the PMA hSiaPsyn cassette flanked by XhoI sites using the primers: GGCTCGAGATTTAAATGCGTACCTCTTCTACGAGATTC (pPMA for XhoI) (SEQ ID NO: 81) and CCCTCGAGATTTAAATCCAACCGATAAGGTGTACAGGAG (PMAtt rev XhoI) (SEQ ID NO: 82). The resulting vector was designated pSH315.

The 2.6 Kb XhoI fragment from pSH315, containing the PMA hSiaPsyn cassette, was ligated into the XhoI site of pSH284 to give the double expression cassette vector pSH321b (FIG. 28).

Generation of pJN711b for Expressing Genes Involved in Making Galactosylated Glycoproteins

The D. melanogaster gene encoding the UDP Galactose Transporter (Genbank Accession No. BAB62747) (referred to as DmUGT) was PCR amplified from a D. melanogaster cDNA library (UC Berkeley Drosophila Genome Project, ovary λ-ZAP library GM), cloned into the pCR2.1 PCR cloning vector (Invitrogen) and sequenced. Primers DmUGT-5′ (5′-GGCTCGAGCGGCCGCCACCATGAATAGCATACACATGAACGCCAATAC G-3′) (SEQ ID NO: 83) and DmUGT-3′ (5′-CCCTCGAGTTAATTAACTAGACGCGCGGCAGCAGCTTCTCCTCATCG-3′) (SEQ ID NO: 84) were used to amplify the gene, which introduced NotI and PacI sites at the 5′ and 3′ ends, respectively. The NotI and PacI sites were then used to subclone DmUGT fused downstream of the PpOCH1 and promoter at the Nod/PacI sites in pRCD393 to create plasmid pSH263. See Davidson et al., WO05/100584A2, which is incorporated by reference herein.

The S. pombe gene encoding UDP-galactose 4-epimerase (GenBank Accession No. ATCC24843) (referred to as SpGALE) was amplified from S. pombe genomic DNA using primers GALE2-L (5′ ATG ACT GGT GTT CAT GAA GGG 3′) (SEQ ID NO: 85) and GALE2-R (5′ TTA CTT ATA TGT CTT GGT ATG 3′) (SEQ ID NO: 86). The amplified product was cloned into pCR2.1 (Invitrogen) and sequenced. Sequencing revealed the presence of an intron (175 bp) at the +66 position. To eliminate the intron, upstream primer GD1 (94 bases) was designed. It has a NotI site, 66 bases upstream of the intron, followed by 20 bases preceding the intron. GD2 is the downstream primer and has a PacI site. Primers GD1 (5′ GCG GCC GCA TGA CTG GTG TTC ATG AAG GGA CTG TGT TGG TTA CTG GCG GCG CTG GTT ATA TAG GTT CTC ATA CGT GCG TTG TFT TGT TAG AAA A 3′) (SEQ ID NO: 87) and GD2 (5′ TTA ATT AAT TAC TTA TAT GTC TTG GTA TG 3′) (SEQ ID NO: 88) were used to amplify the SpGALE intronless gene from the pCR2.1 subclone and the product cloned again into pCR2.1 and sequenced.

The H. sapiens β-1,4-galactosyltransferase I gene (hGalTI, Genbank Accession No. AH003575) was PCR amplified from human kidney cDNA (Marathon-Ready cDNA, Clontech) using primers RCD 192 (5′-GCCGCGACCTGAGCCGCCTGCCCCAAC-3′) (SEQ ID NO: 89) and RCD186 (5′-CTAGCTCGGTGTCCCGATGTCCACTGT-3′) (SEQ ID NO: 90). This PCR product was cloned into pCR2.1 vector (Invitrogen, Carlsbad, Calif.) and sequenced. From this clone, a PCR overlap mutagenesis was performed for three purposes: 1) to remove a NotI site within the open reading frame while maintaining the wild-type protein sequence; 2) to truncate the protein immediately downstream of the endogenous transmembrane domain; and 3) to introduce AscI and PacI sites at the 5′ and 3′ ends for modular cloning. To do this, the 5′ end of the gene up to the NotI site was amplified using primers RCD198 (5′-CTTAGGCGCGCCGGCCGCGACCTGAGCCGCCTGCCC-3′) (SEQ ID NO: 91) and RCD201 (5′-GGGGCATATCTGCCGCCCATC-3′) (SEQ ID NO: 92) and the 3′ end was amplified with primers RCD200 (5′-GATGGGCGGCAGATATGCCCC-3′) (SEQ ID NO: 93) and RCD199 (5′-CTTCTTAATTAACTAGCTCGGTGTCCCGATGTCCAC-3′) (SEQ ID NO: 94). The products were overlapped together with primers 198 and 199 to resynthesize the ORF with the wild-type amino acid sequence while eliminating the NotI site. The new truncated hGalTI PCR product was cloned into pCR2.1 vector (Invitrogen, Carlsbad, Calif.) and sequenced. The introduced AscI/PacI sites were then used to subclone the fragment into plasmid pRCD259, which is a PpURA3/HYGR roll-in vector, to create pRCD260. A library of yeast targeting sequence transmembrane domains as described herein, was ligated into the NotI/AscI sites located upstream of the hGalTI gene to create plasmids pXB20-pXB67. pXB53 is a truncated S. cerevisiae Mnn2 (s) targeting peptide (1-108 nucleotides of MNN2 from Genbank Accession No. NP_(—)009571) ligated in-frame to a 43 N-terminal amino acid deletion of a human β1,4-galactosyltransferase I (Genbank Accession No. AH003575).

Using the above vectors, pJN711b (FIG. 29) was constructed, which is a HYGR plasmid containing hGalTI-53, POCH1-SpGALE, and DmUGT.

Generation of pSH326b for Expressing Human CMP-Sialic Acid Synthase (hCMP-Sia syn) and Mouse CMP-Sialic Acid Transporter (mCMP-Sia Tr)

The gene encoding the human CMP-Sialic acid synthase (Genbank Accession No. AF397212) (referred to as hCMP-Sia syn) was amplified as a NotI-PacI fragment from human prostate cDNA. The conditions used for thermocycling were as follows: 95° C. 2 min, 1 cycle; 97° C. 30 sec, 60° C. 30 sec, 72° C. 2.5 min, 25 cycles; 72° C. 5 min. using oligonucleotide primers: GGGAGAATGCGGCCGCCACCATGGACTCGGTGGAGAAGGGGGCCGCC ACCTC (hCMP-NANA syn NotI/Koz) (SEQ ID NO: 95) and CCTTAATTAACTATTTTTGGCATGAATTATTAACTTTTTCCATTA (hCMP-NANA syn PacI) (SEQ ID NO: 96) with Advantage™ DNA polymerase (BD Biosciences). The resulting 1.3 Kb fragment was cloned into pCR2.1 (Invitrogen), sequenced and designated pHW6. The hCMP-Sia syn was cloned from pHW6 into pPB140 (a Kanamycin resistance vector, containing a 1.2 Kb fragment of P. pastoris HIS3 loci to facilitate ‘rolling-in’ integration, and the GAPDH-CYC promoter-terminator expression cassette) as a NotI-PacI fragment, giving the vector pSH301.

The gene encoding the mouse CMP-Sialic acid transporter (Genbank Accession No. Z71268) (referred to as mCMP-Sia Tr) was amplified as above (using 2.5 min extension time) from mouse brain cDNA using oligonucleotide primers: CGGAATTCCACCATGGCTCCGGCGAGAGAAAATGTCAG (mCMP-NANA trans Koz/for) (SEQ ID NO: 97) and CGGAATTCTCACACACCAATGATTCTCTCTTTTGAAG (mCMP-NANA trans rev) (SEQ ID NO: 98). The resulting fragment was cloned into pCR2.1 (Invitrogen), sequenced and designated pSH194. The mCMP-Sia syn was digested from pSH194 with EcoRI and blunted with T4 DNA polymerase prior to subcloning into pJN664, previously digested with NotI-PacI and blunted, giving the vector pSH306. This construct was used as template to amplify the PMA mCMP-Sia cassette flanked by XhoI sites using the primers: GGCTCGAGATTTAAATGCGTACCTCTTCTACGAGATTC (pPMA for XhoI) (SEQ ID NO: 99) and CCCTCGAGATTTAAATCCAACCGATAAGGTGTACAGGAG (PMAtt rev XhoI) (SEQ ID NO: 100). The resulting vector was designated pSH317.

The 2.6 Kb XhoI fragment from pSH317, containing the PMA mCMP-Sia transporter cassette, was ligated into the XhoI site of pSH301 (containing hCMP-Sia syn) to give the double expression cassette vector pSH326b (FIG. 30).

Generation of pSH370 for Expressing Rat ST6Gal

An N-terminal deletion of the rat ST6Gal (Genbank Accession No. M18769) was amplified from rat liver cDNA using the conditions described above for the hGNE, but using a 2 min extension time instead of 5 min. The primers were: GGCGCGCCAGCAAGCAAGACCCTAAGGAAGACATTCC (rST6GalI d63 AscI) (SEQ ID NO: 101) and CCTTAATTAATCAACAACGAATGTTCCGGAAGCCAGAAAGG (rST6GalI Pace (SEQ ID NO: 102). The resulting fragment was cloned into pCR2.1 (Invitrogen), sequenced and designated pSH271. Subsequently, the 1 kb fragment containing the encoded rST6Gal catalytic domain was subcloned into a nourseothricin selectable vector (Hansen, 2003) generating a fusion of the rST6Gal to the first 108 base pairs of Mnn2 (leader 53), under the control of the PMA promoter. The resulting vector was designated pSH370 (FIG. 31).

Generation of pSH373 for Expressing Mannosidase II (MannII) and GnTII

D. melanogaster mannosidase II (Genbank Accession No. X77652) fused to leader 53 (KD53) was subcloned as a NotI-PacI fragment from pKD53 into the expression vector pJN702. The resulting vector was designated pSH368.

The rat GnTII (Genbank Accession No. U21662) fused to the first 108 base pairs of Mnn2 (leader 53) was digested from pTC53 (see Hamilton, 2003) as a NotI-PacI fragment and subcloned into pJN664 digested with the same enzymes. The resulting vector was designated pSH327.

The 2.7 kb SwaI fragment of pSH327, containing the PMA GnTII expression cassette, was ligated into the PmeI site of pSH368. The resulting vector was designated pSH373 (FIG. 32).

Generation of pSH568 for Expressing Codon-Optimized Human ST6Gal

A codon-optimized version of an N-terminal deletion of human ST6Gal (Genbank Accession No. NM_(—)003032) fused to the first 108 base pairs of Mnn2 (leader 53) was generated by GENEART GmbH (Regensburg Germany). The nucleic acid and amino acid sequences of this codon optimized hST6Gal are shown in FIG. 33. The resulting NotI-PacI fragment was subcloned into the expression vector pJN703b, giving the construct pSH568 (FIG. 34).

pSH568 was digested with SfiI and transformed into the strain YSH272, which was generated in a similar fashion to YSH160, except that the introduction of rST6Gal was omitted and the vector pSH505 was used instead of pSH373. In vector pSH505, the MannII and GntII genes were introduced as leader 4 and leader 5 fusions, respectively. Leader 4 comprises S. cerevisiae mns1s leader, and leader 5 comprises S. cerevisiae mns1m, respectively. See US 2002/0137134.

Engineering In Vivo Transfer of Sialic Acid To N-Glycans in Yeast

P. pastoris YSH1 (Hamilton, 2003) was used as the initial host strain to engineer the glycosylation machinery necessary to produce sialylated glycoproteins. YSH1 (Ura3-) was transformed with pSH321 (hGNE, hSiaPsyn, URA3; described above) digested with ApaI, and a positive clone designated YSH103 was identified using Ura3 as the selectable marker. YSH103 was then transformed with SfiI digested pJN711b (hGalTI-53, POCH1-SpGALE, DmUGT, Δhis1::Hyg; described above), using Hyg as the selectable marker. A transformant designated YSH116 (Δhis1) was selected and transformed with BspEI digested pSH326 (hCMP-Sia syn, mCMP-Sia Tr; described above), using Kan as the selectable marker. A transformant designated YSH125 (Δhis1) was selected and transformed with pSH370 (rST6Gal; described above) digested with EcoRI and a positive clone selected, using Nat as a selectable marker (Hansen, 2003). A transformant designated as YSH145 (Δhis1) was selected and subsequently transformed with SfiI digested pSH373 (MannII, GnTII, (Δarg1::His1); described above), using this as a selectable marker. The resulting transformant designated as YSH160 (66 arg1) produced glycoproteins exhibiting the oligosaccharide structure: NANA₂Gal₂GlcNAc₂Man₃GlcNAc₂ as shown in FIG. 36.

Alternatively, YSH125 (Δhis1) was selected and transformed with pSH568 (codon optimized hST6Gal) digested with SfiI and a positive clone selected using Nat as a selectable marker. A transformant was selected and subsequently transformed with SfiI digested pSH505 (MannII, GnTII, (Δarg1::His1); described above). The resulting transformant designated as YSH272 (Δarg1) produced glycoproteins exhibiting the oligosaccharide structure: NANA₂Gal₂GlcNAc₂Man₃GlcNAc₂ as shown in FIG. 37.

All of the yeast strains were transformed by electroporation (as recommended by the manufacturer of the electroporator BioRad). Integration into the host genome was confirmed by PCR (Nett, 2005).

Glycan Analysis

Oligosaccharides on a recombinant Kringle 3 glycoprotein expressed in each of the strains described above were analyzed using MALDI-TOF MS.

Glycan analysis of glycoproteins produced in strain YSH126, in which strain YSH116 is further transformed with pSH326, showed mass consistent with the glycan structure GalGlcNAcMan₅GlcNAc₂ confirming the in vivo transfer of a terminal galactose residue (FIG. 35).

Glycan analysis of glycoproteins produced in strain YSH145 showed mass consistent with the glycan structure NANAGalGlcNAcMan₅GlcNAc₂ confirming the in vivo transfer of sialic acid onto at least one oligosaccharide branch (FIGS. 35 and 36).

Glycan analysis of glycoproteins produced in strain YSH160 showed mass consistent with the glycan structure NANA₂Gal₂GlcNAc₂Man₃GlcNAc₂ confirming the in vivo transfer of sialic acid onto at least one oligosaccharide branch (FIG. 36).

Glycan analysis of glycoproteins produced in strain YSH272 showed mass consistent with the glycan structure NANA₂Gal₂GlcNAc₂Man₃GlcNAc₂ confirming the in vivo transfer of sialic acid onto at least one oligosaccharide branch (FIG. 37).

Table 13 summarizes the extent of in vivo sialic acid transfer onto a recombinant glycoprotein in an engineered P. pastoris strain made according to the invention disclosed herein. Values represent the percentages of total glycans that contained sialic acid; and thus sensitive to sialidase treatment.

TABLE 13 In Vivo Sialic Acid Transfer In Yeast Strain Mono-sialylated Bi-sialylated Total transfer YSH145 15% — 15% YSH160 20%  2% 22% YSH272 34% 24% 58%

Materials and Method for the Experiments Described in Example 17

MOPS, sodium cacodylate, manganese chloride, UDP-galactose and CMP-N-acetylneuraminic acid were from Sigma. TFA was from Aldrich. β1,4-galactosyltransferase from bovine milk were from Calbiochem. Protein N-glycosidase F, mannosidases, and oligosaccharides were from Glyko (San Rafael, Calif.). DEAE ToyoPearl resin was from TosoHaas. Metal chelating “HisBind” resin was from Novagen (Madison, Wis.). 96-well lysate-clearing plates were from Promega (Madison, Wis.). Protein-binding 96-well plates were from Millipore (Bedford, Mass.). Salts and buffering agents were from Sigma (St. Louis, Mo.). MALDI matrices were from Aldrich (Milwaukee, Wis.).

Shake-Flask Cultivations

A single colony was picked from an YPD plate (<2 weeks old) containing the strain of interest and inoculated into 10 ml of BMGY media in a 50 ml “Falcon” centrifuge tube. The culture was grown to saturation at 24° C. (approx. 48 hours). The seed culture is transferred into a 500 ml baffled volumetric flask containing 150 ml of BMGY media and grown to OD600 of 5±2 at 24° C. (approx. 18 hours). The growth rate of the cells was determined as the slope of a plot of the natural logarithm of OD600 against time. The cells were harvested from the growth medium (BMGY) by centrifugation at 3000g for 10 minutes, washed with BMMY and suspended in 15 ml of BMMY in a 250 ml baffled volumetric flask. After 24 hours, the expression medium flask is harvested by centrifugation (3000g for 10 minutes) and the supernatant analyzed for K3 production.

Bioreactor Cultivations

A 500 ml baffled volumetric flask with 150 ml of BMGY media was inoculated with 1 ml of seed culture (see flask cultivations). The inoculum was grown to an OD600 of 4-6 at 24° C. (approx 18 hours). The cells from the inoculum culture was then centrifuged and resuspended into 50 ml of fermentation media (per litre of media: CaSO4.2.H2O 0.30 g, K2SO4 6.00 g, MgSO4.7H2O 5.00 g, Glycerol 40.0 g, PTM1 salts 2.0 ml, Biotin 4×10-3 g, H3PO4 (85%) 30 ml, PTM1 salts per litre: CuSO4.H2O 6.00, NaI 0.08 g, MnSO4.7H2O 3.00 g, NaMoO4.2H2O 0.20 g, H3BO3 0.02 g, CoCl2.6H2O 0.50 g, ZnCl2 20.0 g, FeSO4.7H2O 65.0 g, Biotin 0.20 g, H2SO4 (98%) 5.00 ml).

Fermentations were conducted in 3 liter dished bottom (1.5 liter initial charge volume) Applikon bioreactors. The fermentors were run in a fed-batch mode at a temperature of 24° C., and the pH was controlled at 4.5±0.1 using 30% ammonium hydroxide. The dissolved oxygen was maintained above 40% relative to saturation with air at 1 atm by adjusting agitation rate (450-900 rpm) and pure oxygen supply. The air flow rate was maintained at 1 vvm. When the initial glycerol (40 g/l) in the batch phase is depleted, which is indicated by an increase of DO, a 50% glycerol solution containing 12 ml/l of PTM1 salts was fed at a feed rate of 12 ml/l/h until the desired biomass concentration was reached. After a half an hour starvation phase, the methanol feed (100% Methanol with 12 ml/l PTM1) is initiated. The methanol feed rate is used to control the methanol concentration in the fermentor between 0.2 and 0.5%. The methanol concentration is measured online using a TGS gas sensor (TGS822 from Figaro Engineering Inc.) located in the offgass from the fermentor. The fermentors were sampled every eight hours and analyzed for biomass (OD₆₀₀, wet cell weight and cell counts), residual carbon source level (glycerol and methanol by HPLC using Aminex 87H) and extracellular protein content (by SDS page, and Bio-Rad protein assay).

Reporter Protein Expression, Purification and Release of N-Linked Glycans

The K3 domain, under the control of the alcohol oxidase 1 (AOX1) promoter, was used as a model protein and was purified using the 6× Histidine tag as reported previously (Choi, 2003). The glycans were released and separated from the glycoproteins by a modification of a previously reported method (Papac and Briggs 1998). After the proteins were reduced and carboxymethylated, and the membranes blocked, the wells were washed three time with water. The protein was deglycosylated by the addition of 30 μl of 10 mM NH4HCO3 pH 8.3 containing one milliunit of N-glycanase (Glyko). After 16 hr at 37° C., the solution containing the glycans was removed by centrifugation and evaporated to dryness.

Protein Purification

Kringle 3 was purified using a 96-well format on a Beckman BioMek 2000 sample-handling robot (Beckman/Coulter Ranch Cucamonga, Calif.). Kringle 3 was purified from expression media using a C-terminal hexa-histidine tag. The robotic purification is an adaptation of the protocol provided by Novagen for their HisBind resin. Briefly, a 150 uL (μL) settled volume of resin is poured into the wells of a 96-well lysate-binding plate, washed with 3 volumes of water and charged with 5 volumes of 50 mM NiSO4 and washed with 3 volumes of binding buffer (5 mM imidazole, 0.5M NaCl, 20 mM Tris-HCL pH7.9). The protein expression media is diluted 3:2, media/PBS (60 mM PO4, 16 mM KCl, 822 mM NaCl pH7.4) and loaded onto the columns. After draining, the columns are washed with 10 volumes of binding buffer and 6 volumes of wash buffer (30 mM imidazole, 0.5M NaCl, 20 mM Tris-HCl pH7.9) and the protein is eluted with 6 volumes of elution buffer (1M imidazole, 0.5M NaCl, 20 mM Tris-HCl pH7.9). The eluted glycoproteins are evaporated to dryness by lyophilyzation.

Release of N-Linked Glycans

The glycans are released and separated from the glycoproteins by a modification of a previously reported method (Papac, et al. A. J. S. (1998) Glycobiology 8, 445-454). The wells of a 96-well MultiScreen IP (Immobilon-P membrane) plate (Millipore) are wetted with 100 uL of methanol, washed with 3×150 uL of water and 50 uL of RCM buffer (8M urea, 360 mM Tris, 3.2 mM EDTA pH8.6), draining with gentle vacuum after each addition. The dried protein samples are dissolved in 30 uL of RCM buffer and transferred to the wells containing 10 uL of RCM buffer. The wells are drained and washed twice with RCM buffer. The proteins are reduced by addition of 60 uL of 0.1M DTT in RCM buffer for 1 hr at 37° C. The wells are washed three times with 300 uL of water and carboxymethylated by addition of 60 uL of 0.1M iodoacetic acid for 30 min in the dark at room temperature. The wells are again washed three times with water and the membranes blocked by the addition of 100 uL of 1% PVP 360 in water for 1 hr at room temperature. The wells are drained and washed three times with 300 uL of water and deglycosylated by the addition of 30 uL of 10 mM NH4HCO3 pH 8.3 containing one milliunit of N-glycanase (Glyko). After 16 hours at 37° C., the solution containing the glycans was removed by centrifugation and evaporated to dryness.

Miscellaneous

Proteins were separated by SDS/PAGE according to Laemmli (Laemmli 1970).

Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry

Molecular weights of the glycans were determined using a Voyager DE PRO linear MALDI-TOF (Applied Biosciences) mass spectrometer using delayed extraction. The dried glycans from each well were dissolved in 15 uL of water and 0.5 uL spotted on stainless steel sample plates and mixed with 0.5 uL of S-DHB matrix (9 mg/mL of dihydroxybenzoic acid, 1 mg/mL of 5-methoxysalicilic acid in 1:1 water/acetonitrile 0.1% TFA) and allowed to dry.

Ions were generated by irradiation with a pulsed nitrogen laser (337 nm) with a 4 ns pulse time. The instrument was operated in the delayed extraction mode with a 125 ns delay and an accelerating voltage of 20 kV. The grid voltage was 93.00%, guide wire voltage was 0.10%, the internal pressure was less than 5×10-7 torr, and the low mass gate was 875 Da. Spectra were generated from the sum of 100-200 laser pulses and acquired with a 2 GHz digitizer. Man5GlcNAc2 oligosaccharide was used as an external molecular weight standard. All spectra were generated with the instrument in the positive ion mode. The estimated mass accuracy of the spectra was 0.5%.

Example 18 Engineering K. lactis Cells to Produce N-Glycans with the Structure Man₅GlcNAc₂

Identification and Disruption of the K. lactis OCH1 Gene

The OCH1 gene of the budding yeast S. cerevisiae encodes a 1,6-mannosyltransferase that is responsible for the first Golgi localized mannose addition to the Man₈GlcNAc₂ N-glycan structure on secreted proteins (Nakanishi-Shindo, 1993). This mannose transfer is generally recognized as the key initial step in the fungal specific polymannosylation of N-glycan structures (Nakanishi-Shindo et al., 1993; Nakayama, 1992; Morin-Ganet, 2000). Deletion of this gene in S. cerevisiae results in a significantly shorter N-glycan structure that does not include this typical polymannosylation or a growth defect at elevated temperatures (Nakayama, 1992).

The Och1p sequence from S. cerevisiae was aligned with known homologs from Candida albicans (Genbank Accession No. AAL49987), and P. pastoris (Choi, 2003) along with the Hoc1 proteins of S. cerevisiae (Neiman, 1997) and K. lactis (PENDANT EST database) which are related but distinct mannosyltransferases. Regions of high homology that were in common among Och1p homologs but distinct from the Hoc1p homologs were used to design pairs of degenerate primers that were directed against genomic DNA from the K. lactis strain MG1/2 (Bianchi, 1987). PCR amplification with primers RCD33 (CCAGAAGAATTCAATTYTGYCARTGG) (SEQ ID NO:34) and RCD34 (CAGTGAAAATACCTGGNCCNGTCCA) (SEQ ID NO:35) resulted in a 302 bp product that was cloned and sequenced and the predicted translation was shown to have a high degree of homology to Och1 proteins (>55% to S. cerevisiae Och1p).

The 302 bp PCR product was used to probe a Southern blot of genomic DNA from K. lactis strain (MG1/2) with high stringency (Sambrook et al., 1989). Hybridization was observed in a pattern consistent with a single gene indicating that this 302 bp segment corresponds to a portion of the K. lactis genome and K. lactis (KlOCH1) contains a single copy of the gene. To clone the entire KlOCH1 gene, the Southern blot was used to map the genomic locus. Accordingly, a 5.2 kb BamHI/PstI fragment was cloned by digesting genomic DNA and ligating those fragments in the range of 5.2 kb into pUC19 (New England Biolabs, Beverly, Mass.) to create a K. lactis subgenomic library. This subgenomic library was transformed into E. coli and several hundred clones were tested by colony PCR using RCD 33/34. The 5.2 kb clone containing the predicted KlOCH1 gene was sequenced and an open reading frame of 1362 bp encoding a predicted protein that is 46.5% identical to the S. cerevisiae OCH1 gene. The 5.2 kb sequence was used to make primers for construction of an och1::KAN^(R) deletion allele using a PCR overlap method (Davidson, 2002). This deletion allele was transformed into two K. lactis strains and G418 resistant colonies selected. These colonies were screened by both PCR and for temperature sensitivity to obtain a strain deleted for the OCH1 ORF. The results of the experiment show strains which reveal a mutant PCR pattern, which were characterized by analysis of growth at various temperatures and N-glycan carbohydrate analysis of secreted and cell wall proteins following PNGase digestion. The och1 mutation conferred a temperature sensitivity which allowed strains to grow at 30° C. but not at 35° C. FIG. 12A shows a MALDI-TOF analysis of a wild type K. lactis strain producing N-glycans of Man₈GlcNAc₂ [c] and higher.

Identification, Cloning, and Disruption of the K. lactis MNN1 Gene

S. cerevisiae MNN1 is the structural gene for the Golgi α-1,3-mannosyltransferase. The product of MNN1 is a 762-amino acid type II membrane protein (Yip, 1994). Both N-linked and O-linked oligosaccharides isolated from mnn1 mutants lack α-1,3-mannose linkages (Raschke, 1973).

The Mnn1p sequence from S. cerevisiae was used to search the K. lactis translated genomic sequences (PEDANT). One 405 bp DNA sequence encoding a putative protein fragment of significant similarity to Mnn1p was identified. An internal segment of this sequence was subsequently PCR amplified with primers KMN1 (TGCCATCTTTTAGGTCCAGGCCCGTTC) (SEQ ID NO:36) and KMN2 (GATCCCACGACGCATCGTATTTCTTTC), (SEQ ID NO:37) and used to probe a Southern blot of genomic DNA from K. lactis strain (MG1/2). Based on the Southern hybridization data a 4.2 Kb BamHI-PstI fragment was cloned by generating a size-selected library as described herein. A single clone containing the K. lactis MNN1 gene was identified by whole colony PCR using primers KMN1 (SEQ ID NO:36) and KMN2 (SEQ ID NO:37) and sequenced. Within this clone a 2241 bp ORF was identified encoding a predicted protein that was 34% identical to the S. cerevisiae MNN1 gene. Primers were designed for construction of a mnn1::NAT^(R) deletion allele using the PCR overlap method (Davidson, 2002).

This disruption allele was transformed into a strain of K. lactis by electroporation and Noursethoicin resistant transformants were selected and PCR amplified for homologous insertion of the disruption allele. Strains that reveal a mutant PCR pattern may be subjected to N-glycan carbohydrate analysis of a known reporter gene.

FIG. 12B depicts the N-glycans from the K. lactis och1 mnn1 deletion strain observed following PNGase digestion and MALDI-TOF as described herein. The predominant peak at 1908 (m/z) indicated as [d] is consistent with the mass of Man₉GlcNAc₂.

Example 19 Engineering Plant Cells To Express GlcNAc Transferases or Galactosyltransferases

GlcNAc transferase IV is required for the addition of β1,4 GlcNAc to the α-1,6 mannose residue and the α-1,3 mannose residues in complex N-glycans in humans. So far, GlcNAc transferase IV has not been detected in or isolated from plants. A transgenic plant that is capable of adding human-like N-glycans must therefore be engineered to express GlcNAc transferase IV. Thus, the plant host cell or transgenic plant must also localize an expressed GlcNAc transferase IV to the correct intracellular compartment in the host so that the enzyme can add the β1,4 GlcNAc to the appropriate mannose residues.

There is some evidence that glycosyltransferases from mammals and plants have similar targeting signals. For example, a full-length rat α-2,6-sialyltransferase has been shown to correctly localize to the trans Golgi network in transgenic Arabidopsis plant cells, but was not necessarily active there (Wee, 1998). A fusion construct having fifty-two N-terminal amino acids from α-2,6-sialyltransferase fused to a green fluorescent reporter protein (GFP) was also shown to correctly localize to the plant Golgi (Boevink, 1998). Two mammalian proteins—TGN30 and furin—and AtELP, an Arabidopsis integral membrane protein (Sanderfoot, 1998), which localize to the trans Golgi network, each contain a tyrosine tetrapeptide motif which targets them to the Golgi, probably by a recycling mechanism via the plasma membrane. Although mammals and plants appear to share some common mechanisms related to protein targeting, exogenous glycosylases may nonetheless not target correctly in a plant cell, however, localization does not necessarily equate with enzyme activity. It therefore becomes essential to devise means to correctly target in a plant cell these enzymes and/or other enzymes that participate in forming complex, human-like N-glycans.

Glycosylation enzymes are integral membrane proteins which reside in the endoplasmic reticulum and Golgi apparatus. The targeting and localization signals are normally contained in the cytoplasmic and/or transmembrane domains and in some cases are contained in some lumenal domains. For example, fifty-two amino acids that make up the transmembrane domain, nine cytoplasmic amino acids and twenty-six lumenal amino acids of α-2,6-sialyltransferase are required to target GFP to the trans Golgi network (Boevink, 1998).

Thus, a library of sequences encoding cellular targeting signal peptides comprising either just the cytoplasmic and transmembrane domains or the cytoplasmic, transmembrane and lumenal domains of endoplasmic reticulum and Golgi specific proteins is generated, as described in Example 11. The targeting peptide sequences may be chosen from ER and Golgi-resident plant, yeast or animal proteins. A glycosylation related protein, e.g., an enzyme (or catalytic domain thereof) such as a glycosylase or integral membrane enzyme can be fused in-frame to the library of targeting peptide sequences and introduced into plants (FIG. 13). Plant targeting peptide sequences may be most efficient in localizing the chimeric enzymes to the ER and Golgi, although targeting peptide sequences from fungi and mammals may also be effective. For example, the N-terminal 77 amino acids from tobacco N-acetylglucosaminyl Transferase I have been shown to correctly target a reporter protein to the Golgi (Essl, 1999). In one embodiment, one or more N-terminal fragments comprising these 77 amino acids (or subsets of these amino acids) is fused to one or more fragments comprising a catalytic domain of GlcNAc transferase IV. At least one resulting fusion protein correctly localizes a functional GlcNAc transferase IV to the Golgi apparatus in a plant cell, as evidenced by monitoring the glycosylation state of a reporter glycoprotein resident or introduced into the plant host cell using techniques described herein.

Another plant enzyme shown to localize to the Golgi is Arabidopsis GlcNAc transferase II (Strasser, 1999). Thus, in another embodiment, one or more different fragments of the Arabidopsis GlcNAc transferase II targeting peptide are fused to a GlcNAc transferase IV catalytic domain and fusion constructs produced and tested as described above. The plant specific β1,2-xylosyltransferase from Arabidopsis thaliana is another protein that localizes to the Golgi and its localization and retention in the Golgi is dependent on its cytoplasmic and transmembrane sequences (Dimberger, 2002). Thus, in another embodiment, one or more fragments comprising the cytoplasmic and transmembrane sequences of β1,2-xylosyltransferase are fused to one or more fragments comprising a GlcNAc transferase IV catalytic domain and resulting fusion constructs are transformed into plant cells and tested for their ability to produce a human-like N-glycan and to otherwise modulate glycosylation in the plant host cell.

Because GlcNAc transferase IV or Galactosyltransferase from one organism may function more efficiently in a specific plant host than one from another organism, fragments comprising GlcNAc transferase IVs (or catalytic domains) from various eukaryotic organisms are fused in-frame to the library of endoplasmic reticulum (ER) and Golgi targeting peptide sequences and are then introduced into plants. The use of a library of nucleic acids encoding enzyme domains isolated or derived from different species increases the chances of efficient glycosylation—in addition to correct localization and glycosylation by GlcNAc transferase IV.

The methods and combinatorial nucleic acid libraries of the invention may be used to introduce and localize, sequentially or en masse, multiple enzymes required to glycosylate proteins in a plant cell with human-like N-glycans. As different plant species may require different growth conditions, protocols for transformation may vary depending on the species being transformed (Potrykus, 1990). The commonly used methods for generating transgenic plants include Agrobacterium mediated transformation, particle bombardment (Sanford, 1990) and electroporation.

Agrobacterium Method

The catalytic domains of GlcNAc transferase IVs are fused in-frame to multiple different targeting peptide sequences known to target proteins to the ER and Golgi in plants. Each of these fusion constructs is introduced under the control of the ubiquitously expressed promoters like the 35S CaMV, ubiquitin or actin promoters, tissue specific promoters or inducible promoters. A plant specific terminator region is also used. This cassette (promoter::targeting peptide-GlcNAc transferase IV::terminator) is cloned into a vector suitable for Agrobacterium mediated transformation (FIG. 13). The vector also contains a selectable marker that allows one to select for transformed plants. The common selectable markers used include those resulting in kanamycin, hygromycin and basta resistance. The construct is introduced into Agrobacterium via well-established transformation methods, which are available in the art. An Agrobacterium library of Golgi-targeted GlcNAc transferase IVs is thereby generated.

Embryonic and meristematic tissue may be transformed and can regenerate transgenic plants. To transform tissue, tissue explants (these could be plumules and radicals from germinated seeds) are first soaked and coated with an Agrobacterium innoculum. They are then cultured on plates containing the innoculum to form an undifferentiated mass of cells termed the callus. Transformed plant cells are selected for by adding to the medium the relevant kanamycin, hygromycin or basta (depending on the selectable marker used on the construct). The transformed plant cells can either be grown in culture and remain undifferentiated or they are treated with shoot regenerating and shoot elongation medium. Explants that differentiate are transferred onto rooting medium to generate transgenic plants. Some plants like Arabidopsis can be transformed by dipping flowers into an Agrobacterium solution. Seeds from the transformed plants are germinated on plates containing the relevant herbicide or antibiotic selection. Transgenic plants are those that grow on the selection media. The transgenic plants are then screened for those with properly glycosylated proteins (i.e., those which have complex, human-like N-glycans) by isolating glycoproteins from plant extracts and analyzing glycoprotein patterns as described elsewhere herein, e.g., by using a specific antibody or lectin. Although the Agrobacterium method is economical and simple, it is limited to certain species of plants. Accordingly, plants that cannot be transformed using Agrobacterium can be transformed by ballistics or electroporation.

Particle Bombardment Method and Electroporation

Compared to Agrobacterium mediated transformation, these methods have a greater tendency to insert multiple copies of the transgene into the genome. This could result in gene silencing and cosuppression. However, unlike Agrobacterium mediated transformation, these methods are not species limited and are therefore useful when an Agrobacterium method cannot be employed to generate transgenic plants. In the particle bombardment method, cultured plant cells are bombarded with very small tungsten or gold particle that have been coated with DNA (promoter::targeting peptide-GlcNAc transferase IV-terminator::selectable marker) (FIG. 13) (rb and lb not required) while in the electroporation method, plant cells in a DNA (promoter::targeting peptide-GlcNAc transferase IV-terminator::selectable marker) solution are treated with an electric pulse that perforates the cell, allowing it to take up DNA. The cells are then cultured and allowed to recover. Stable transformants are selected for by culturing and regenerating plants on appropriate selection medium.

Engineering Soybeans to Express GlcNAc Transferase IV Using a Soybean Cotyledonary Node Agrobacterium Mediated Transformation System

An Agrobacterium library of Golgi-targeted GlcNAc transferase IV is generated as described above. Soybean explants are transformed with the library using a protocol described by Hinchee, 1988. A reporter protein is expressed with a His tag, purified and then analyzed. Transgenic plants are assayed for proteins with the α-1,6 mannose and the α-1,3 mannose residues using, e.g., mass spectroscopy.

Engineering Pea to Express GlcNAc Transferase IV Using Particle Bombardment

A GlcNAc transferase IV plasmid library is coated onto tungsten or gold particles and used as microprojectiles to bombard calli derived from pea embryonic tissue as described (Molnar, 1999). A reporter protein is expressed with a His tag, purified and then analyzed. Transgenic plants are assayed for proteins with the α-1,6 mannose and the α-1,3 mannose residues using, e.g., MALDI.

Engineering Plants to Express GlcNAc Transferase I

GlcNAc transferase I is involved in the addition of GlcNAc to the terminal α-1,3 mannose residue to form Man₅GlcNAc₂, an essential step in the maturation of complex N-glycans. Although GlcNAc transferase I has been isolated from plants and appears to have the same function as its mammalian homolog, it may not be the most efficient enzyme for glycosylation of mammalian or exogenous proteins and may not be found in every plant species. As the addition of GlcNAc to the terminal α-1,3 mannose residue is a controlling step in the mammalian glycosylation pathway, it is advantageous to have transgenic plants that can carry out this step efficiently. To create transgenic plants that express GlcNAc transferase I that can function efficiently to promote the formation of complex N-glycans, a library of GlcNAc transferase I isolated or derived from various organisms is fused in-frame to multiple plant Golgi targeting peptide sequences according to the methods described herein. The combinatorial library thus created is introduced into a plant cell or organism as described above for GlcNAc transferase IV.

Engineering Maize to Express GlcNAc Transferase I Using Particle Bombardment

Transgenic maize can be obtained using a protocol similar to the one used to generate peas that express GlcNAc transferase IV. Here the GlcNAc transferase I plasmid library is coated onto tungsten or gold particles and used to bombard calli derived from maize embryonic tissue, e.g., using a protocol specific for the generation of transgenic maize (Gordon-Kamm, 1990). Transgenic plants are assayed for proteins having GlcNAc on the terminal α-1,3 mannose residue, e.g., using specific antibodies or by assaying reduced binding of the N-glycans to certain lectins or by using MALDI-TOF.

Other useful references for using plant host cells according to the invention include: Dirnberger, 2002; Frame, 2002; Gomord, 1999; Laursen, 1994; Orci, 2000; Newell, 2002; Pawlowski, 1996; Schroeder, 1993; Sorokin, 2000; Strasser, 1999; Tomes, 1990.

Engineering Plant Cells to Produce β1,4-Galactosyltransferases

β1,4-galactosyltransferase is an important human glycosyltransferase that is absent in plants. Lerouge, 1998. In mammals, β1,4-galactosyltransferase is localized in the Golgi and is responsible for the transfer of galactose residues to the terminal N-acetylglucosamine of the core Man₃GlcNAc₂ of complex N-glycans. In plants, the Man₃GlcNAc₂ core contains β1,2-xylose and α1,3-fucose residues and lacks the β1,4-galactose. The xylose and fucose modifications are implicated in allergies and act as antigenic epitopes and are therefore not desirable modifications of therapeutic proteins.

The galactose modifications carried out by β1,4-galactosyltransferase can be important for the proper functioning of the therapeutic proteins. In mammals, β1,4-galactosyltransferase acts after N-acetylglucosaminyltransferase I and N-acetylglucosaminyltransferase II and has been shown to initiate branching of the complex N-glycan. Lerouge, 1998; Palacpac, 1999. In tobacco cells, expression of human β1,4-galactosyltransferase has been shown to result in galactosylated N-glycans with reduced fucose and xylose modifications. Bakker, 2001; Fujiyama, 2001; Palacpac, 1999. In these studies, a 1.2 kb fragment of human β1,4-galactosyltransferase was cloned downstream of the cauliflower mosaic virus promoter (35SCaMV), introduced into the binary vector pGA482, and finally into tobacco cells. Palacpac, 1999.

Tobacco cells were transformed using the agrobacterium method described by Rempel et al. (Rempel, 1995). Transformation of tobacco cells has also been described (An, 1985). Expression of β1,4-galactosyltransferase under the 35SCaMV resulted in ubiquitous expression of the gene in tobacco cells. Tobacco cells expressing human β1,4-galactosyltransferase showed the presence of galactosylated N-glycans. (Palacpac, 1999; Bakker 1991), showed that crossing tobacco plants expressing human β1,4-galactosyltransferase with plants expressing the heavy and light chain of a mouse antibody resulted in plants in which the antibody showed 30% galactosylation (Bakker, 2001).

A combinatorial DNA library can be constructed to obtain a β1,4-galactosyltransferase plant cell line for the addition of galactose residues. The combinatorial DNA library can effectively produce cell lines which are more efficient in the addition of galactose residues. Once such a cell line is made it can be easily crossed to cell lines expressing other glycosylation enzymes and to those expressing therapeutic proteins to produce therapeutic proteins with human-like glycosylation. The final line can then be grown as plants and harvested to extract proteins or can be cultured as plant cells in suspension cultures to produce proteins in bioreactors. By expressing the therapeutic proteins using the library of signal peptides, it is possible to retain the therapeutic protein within the cells or have them secreted into the medium. Tobacco cells expressing β1,4-galactosyltransferase secrete galactosylated N-glycans (Ryo, 2002). While horseradish peroxidase isozyme C expressed in tobacco plants expressing β1,4-galactosyltransferase contained xylose and fucose modifications, no xylose or fucose modification could be detected in horseradish peroxidase isozyme C expressed in tobacco cells expressing β1,4-galactosyltransferase (GT6 cells). (Fujiyama, 2001). This indicates that it may be advantageous to express therapeutic proteins in cell lines instead of whole plants.

Engineering Plants to Produce Sialyltransferase

In mammals, sialyltransferase is a trans Golgi enzyme that adds terminal sialic acid residues to glycosylated polypeptides. Thus far, terminal sialic acid residues have not been detected in plants (Wee, 1998). Wee et al. expressed the rat α-2,6-sialyltransferase in transgenic Arabidopsis and showed that the enzyme properly localized to the Golgi and was functional. Wee et al. demonstrated that membranes isolated from transgenic Arabidopsis, when incubated with CMP-3H-sialic acid and asialofetuin acceptor, resulted in the addition of sialic acid residues while membrane isolated from wild-type Arabidopsis did not. While expressing the rat α-2,6-sialyltransferase in Arabidopsis resulted in a functional enzyme that was able to incorporate sialic acid residues, fusing the mammalian enzymes α-2,3-sialyltransferase and α-2,6-sialyltransferase to a variety of transit peptides using the library approach of the present invention (described above) can result in more efficient sialylation in other plant species. Wee et al. had to isolate membranes and incubate them with CMP-³H-sialic acid and asialofetuin acceptor since Arabidopsis does not have CMP-sialic acid or its transporter. In order to overcome this additional step and obtain sialic acid addition in the plant, CMP-sialic acid biosynthetic pathway and the CMP-sialic acid transporter can be co-expressed in transgenic plants expressing α-2,3-sialyltransferase and α-2,6-sialyltransferase (see Examples 6, 16 and 17). As an alternative, the CMP-sialic acid transporter can be co-expressed with α-2,3-sialyltransferase and α-2,6-sialyltransferase in plant cells grown in suspension culture, and CMP-sialic acid or other precursors of CMP-sialic acid supplied in the medium.

Expressing α-2,3-Sialyltransferase and α-2,6-Sialyltransferase in Lemna

As described in the U.S. Pat. No. 6,040,498 (“the '498 patent”), lemna (duckweed) can be transformed using both agrobacterium and ballistic methods. Using protocols described in the '498 patent, lemna will be transformed with a library of Golgi targeted α-2,3-sialyltransferase and/or α-2,6-sialyltransferase and a library of mammalian CMP-sialic acid transporters. Transgenic plants can be assayed for those that produce proteins with terminal sialic acid residues according to screening techniques discussed herein (Example 17).

Expressing α-2,3-Sialyltransferase and α-2,6-Sialyltransferase in Tobacco Cells

Alpha-2,3-sialyltransferase and/or α-2,6-sialyltransferase and/or a library of mammalian CMP-sialic acid transporters can also be introduced into tobacco cells grown in suspension culture as described for β1,4-galactosyltransferases. CMP-sialic acid can be added to the medium. Both the cells and the culture medium (secreted proteins) can be assayed for proteins with terminal sialic acid residues according to screening techniques discussed herein (Example 17).

Example 20 Engineering Insect Cells to Produce Glycosyltransferases

Insect cells provide another mechanism for producing glycoproteins but the resulting glycoproteins are not complex human-like glycoproteins. (Marz, 1995; Jarvis, 1997.) It is another feature of the present invention to provide enzymes in insect cells, which are targeted to the organelles in the secretory pathway. In a preferred embodiment, enzymes such as glycosyltransferases, galactosyltransferases and sialyltransferases are targeted to the ER, Golgi or the trans Golgi network in lepidopteran insect cells (Sf9). Expression of mammalian β1,4-galactosyltransferase has been shown in Sf9 cells. Hollister, 1998. These enzymes are targeted by means of a chimeric protein comprising a cellular targeting signal peptide not normally associated with the enzyme. The chimeric proteins are made by constructing a nucleic acid library comprising targeting sequences as described herein and the glycosylation enzymes. Baculovirus expression in insect cells is commonly used for stable transformation for adding mammalian glycosyltransferases in insect cells. (Hollister, 2001.)

TABLE 11 DNA And Protein Sequence Resources 1. European Bioinformatics Institute (EBI) (a centre for research and services in bioinformatics) 2. Swissprot database 3. List of known glycosyltransferases and their origin. 4. human cDNA, Kumar et al (1990) Proc. Natl. Acad. Sci. USA 87: 9948-9952 5. human gene, Hull et al (1991) Biochem. Biophys. Res. Commun. 176: 608-615 6. mouse cDNA, Kumar et al (1992) Glycobiology 2: 383-393 7. mouse gene, Pownall et al (1992) Genomics 12: 699-704 8. murine gene (5′ flanking, non-coding), Yang et al (1994) Glycobiology 5: 703-712 9. rabbit cDNA, Sarkar et al (1991) Proc. Natl. Acad. Sci. USA 88: 234-238 10. rat cDNA, Fukada et al (1994) Biosci. Biotechnol. Biochem. 58: 200-201 1,2 (GnTII) EC 2.4.1.143 11. human gene, Tan et al (1995) Eur. J. Biochem. 231: 317-328 12. rat cDNA, D'Agostaro et al (1995) J. Biol. Chem. 270: 15211-15221 13. arabadopsis cDNA Strasser et al (1999) J. Glycoconj. 16: 787-791 14. C. elegans gene Chen et al., (2002) Biochim. Biophys. Acta. 1573: 271-279 β1,4 (GnTIII) EC 2.4.1.144 15. human cDNA, Ihara et al (1993) J. Biochem. 113: 692-698 16. murine gene, Bhaumik et al (1995) Gene 164: 295-300 17. rat cDNA, Nishikawa et al (1992) J. Biol. Chem. 267: 18199-18204 β1,4 (GnTIV) EC 2.4.1.145 18. human cDNA, Yoshida et al (1998) Glycoconjugate Journal 15: 1115-1123 19. bovine cDNA, Minowa et al., European Patent EP 0 905 232 20. β1,6 (GnT V) EC 2.4.1.155 21. human cDNA, Saito et al (1994) Biochem. Biophys. Res. Commun. 198: 318-327 22. rat cDNA, Shoreibah et al (1993) J. Biol. Chem. 268: 15381-15385 β1,4 Galactosyltransferase, EC 2.4.1.90 (LacNAc synthetase) EC 2.4.1.22 (lactose synthetase) 23. bovine cDNA, D'Agostaro et al (1989) Eur. J. Biochem. 183: 211-217 24. bovine cDNA (partial), Narimatsu et al (1986) Proc. Natl. Acad. Sci. USA 83: 4720-4724 25. bovine cDNA (partial), Masibay & Qasba (1989) Proc. Natl. Acad. Sci. USA 86: 5733-5377 26. bovine cDNA (5′ end), Russo et al (1990) J. Biol. Chem. 265: 3324 27. chicken cDNA (partial), Ghosh et al (1992) Biochem. Biophys. Res. Commun. 1215-1222 28. human cDNA, Masri et al (1988) Biochem. Biophys. Res. Commun. 157: 657-663 29. human cDNA, (HeLa cells) Watzele & Berger (1990) Nucl. Acids Res. 18: 7174 30. human cDNA, (partial) Uejima et al (1992) Cancer Res. 52: 6158-6163 31. human cDNA, (carcinoma) Appert et al (1986) Biochem. Biophys. Res. Commun. 139: 163-168 32. human gene, Mengle-Gaw et al (1991) Biochem. Biophys. Res. Commun. 176: 1269-1276 33. murine cDNA, Nakazawa et al (1988) J. Biochem. 104: 165-168 34. murine cDNA, Shaper et al (1988) J. Biol. Chem. 263: 10420-10428 35. murine cDNA (novel), Uehara & Muramatsu unpublished 36. murine gene, Hollis et al (1989) Biochem. Biophys. Res. Commun. 162: 1069-1075 37. rat protein (partial), Bendiak et al (1993) Eur. J. Biochem. 216: 405-417 2,3-Sialyltransferase, (ST3Gal II) (N-linked) (Gal-1,3/4-GlcNAc) EC 2.4.99.6 38. human cDNA, Kitagawa & Paulson (1993) Biochem. Biophys. Res. Commun. 194: 375-382 39. rat cDNA, Wen et al (1992) J. Biol. Chem. 267: 21011-21019 40. murine cDNA Lee et al., (1994) J. Biol. Chem. 269: 10028-10033 2,6-Sialyltransferase, (ST6Gal I) EC 2.4.99.1 41. chicken, Kurosawa et al (1994) Eur. J. Biochem 219: 375-381 42. human cDNA (partial), Lance et al (1989) Biochem. Biophys. Res. Commun. 164: 225-232 43. human cDNA, Grundmann et al (1990) Nucl. Acids Res. 18: 667 44. human cDNA, Zettlmeisl et al (1992) Patent EPO475354-A/3 45. human cDNA, Stamenkovic et al (1990) J. Exp. Med. 172: 641-643 (CD75) 46. human cDNA, Bast et al (1992) J. Cell Biol. 116: 423-435 47. human gene (partial), Wang et al (1993) J. Biol. Chem. 268: 4355-4361 48. human gene (5′ flank), Aasheim et al (1993) Eur. J. Biochem. 213: 467-475 49. human gene (promoter), Aas-Eng et al (1995) Biochim. Biophys. Acta 1261: 166-169 50. mouse cDNA, Hamamoto et al (1993) Bioorg. Med. Chem. 1: 141-145 51. rat cDNA, Weinstein et al (1987) J. Biol. Chem. 262: 17735-17743 52. rat cDNA (transcript fragments), Wang et al (1991) Glycobiology 1: 25-31, Wang et al (1990) J. Biol. Chem. 265: 17849-17853 53. rat cDNA (5′ end), O'Hanlon et al (1989) J. Biol. Chem. 264: 17389-17394; Wang et al (1991) Glycobiology 1: 25-31 54. rat gene (promoter), Svensson et al (1990) J. Biol. Chem. 265: 20863-20688 55. rat mRNA (fragments), Wen et al (1992) J. Biol. Chem. 267: 2512-2518

Additional methods and reagents that can be used in the methods for modifying the glycosylation are described in the literature, such as U.S. Pat. No. 5,955,422, U.S. Pat. No. 4,775,622, U.S. Pat. No. 6,017,743, U.S. Pat. No. 4,925,796, U.S. Pat. No. 5,766,910, U.S. Pat. No. 5,834,251, U.S. Pat. No. 5,910,570, U.S. Pat. No. 5,849,904, U.S. Pat. No. 5,955,347, U.S. Pat. No. 5,962,294, U.S. Pat. No. 5,135,854, U.S. Pat. No. 4,935,349, U.S. Pat. No. 5,707,828, and U.S. Pat. No. 5,047,335. Appropriate yeast expression systems can be obtained from sources such as the American Type Culture Collection, Rockville, Md. Vectors are commercially available from a variety of sources.

Sequence Listings

SEQUENCE LISTINGS SEQ ID NO: 1-6 can be found in U.S. Pat. Application No. 09/892,591 SEQ ID NO: 7 Primer: regions of high homology within 1,6 mannosyltransferases 5′-atggcgaaggcagatggcagt-3′ SEQ ID NO: 8 Primer: regions of high homology within 1,6 mannosyltransferases 5′-ttagtccttccaacttccttc-3′ SEQ ID NO: 9 internal primer: 5′-actgccatctgccttcgccat-3′ SEQ ID NO: 10 internal primer: 5′-GTAATACGACTCACTATAGGGC-3′ T7 SEQ ID NO: 11 Internal primer: 5′-AATTAACCCTCACTAAAGGG-3′ T3 SEQ ID NO: 12 Primer: atgcccgtgg ggggcctgtt gccgctcttc agtagc SEQ ID NO: 13 Primer: tcatttctct ttgccatcaa tttccttctt ctgttcacgg SEQ ID NO: 14 Primer: ggcgcgccga ctcctccaag ctgctcagcg gggtcctgtt ccac SEQ ID NO: 15 Primer: ccttaattaa tcatttctct ttgccatcaa tttccttctt ctgttcacgg SEQ ID NO: 16 Primer: ggcgagctcg gcctacccgg ccaaggctga gatcatttgt ccagcttcaga SEQ ID NO: 17 Primer: gcccacgtcg acggatccgt ttaaacatcg attggagagg ctgacaccgc tacta SEQ ID NO: 18 Primer: cgggatccac tagtatttaa atcatatgtg cgagtgtaca actcttccca catgg SEQ ID NO: 19 Primer: ggacgcgtcg acggcctacc cggccgtacg aggaatttct cggatgactc ttttc SEQ ID NO: 20 Primer: cgggatccct cgagagatct tttttgtaga aatgtcttgg tgcct SEQ ID NO: 21 Primer: ggacatgcat gcactagtgc ggccgccacg tgatagttgt tcaattgatt gaaataggga caa SEQ ID NO: 22 Primer: ccttgctagc ttaattaacc gcggcacgtc cgacggcggc ccacgggtcc ca SEQ ID NO: 23 Primer: ggacatgcat gcggatccct taagagccgg cagcttgcaa attaaagcct tcgagcgtcc c SEQ ID NO: 24 Primer: gaaccacgtc gacggccatt gcggccaaaa ccttttttcc tattcaaaca caaggcattg c SEQ ID NO: 25 Primer: ctccaatact agtcgaagat tatcttctac ggtgcctgga ctc SEQ ID NO: 26 Primer: tggaaggttt aaacaaagct agagtaaaa tagatatagc gagattagag aatg SEQ ID NO: 27 Primer: aagaattcgg ctggaaggcc ttgtaccttg atgtagttcc cgttttcatc SEQ ID NO: 28 Primer: gcccaagccg gccttaaggg atctcctgat gactgactca ctgataataa aaatacgg SEQ ID NO: 29 Primer: gggcgcgta tttaaatacta gtggatctat cgaatctaaa tgtaagttaa aatctctaa SEQ ID NO: 30 Primer: ggccgcctgc agatttaaat gaattcgg cgcgccttaat SEQ ID NO: 31 Primer: taaggcgcgc cgaattcatt taaatctgca gggc SEQ ID NO: 32 Primer: 5′-tggcaggcgcgcctcagtcagcgctctcg-3′ SEQ ID NO: 33 Primer: 5′-aggttaatta agtgctaattccagctagg-3′ SEQ ID NO: 34 primer for K. lactis OCH1 gene: ccagaagaat tcaattytgy cartgg SEQ ID NO: 35 primer for K. lactis OCH1 gene: cagtgaaaat acctggnccn gtcca SEQ ID NO: 36 primer for K. lactis MNN1 gene: tgccatcttt taggtccagg cccgttc SEQ ID NO: 37 primer for K. lactis MNN1 gene: gatcccacga cgcatcgtat ttctttc SEQ ID NO: 38 DNA sequence of the 302 bp segment of the putative KlOCH1 gene: gcccttcagtgaaaatacctggcccggtccagttcataatatcggtaccatctgtatttttggcggttttcttttgttgatgttt gtaatttttgttgaacttctttttatccctcatgttgacattataatcatctgcaatgtcttttaatacttcagc atcatctaaaggaatgctgcttttaacatttgccacgctctccaatgttgttgcggtgatatttgtgatcaattcgcgcaataa tggatggccagattttgattgtattgtccactgacaaaattgaattctctggaagggc SEQ ID NO: 39 Translation of putative KlOCH1 gene (excluding primers): TIQSKSGHPLLRELITNITATTLESVANVKSSIPLDDAEVLKDIADDYNVNM RDKKKFNKNYKHQQKKTAKNTDGTDIMN SEQ ID NO: 40 DNA sequence of the 405 bp segment of the putative KlMNN1 gene: cccagcgtgccattaccgtatttgccgccgtttgaaatactcaatattcatgatggttgtaaggcgttttttatcattcgcgat ataatatgccatcttttaggtccaggcccgttctcttagctatctttggtgtctgtgctaccgtgatatggtacct attctttttccagtctaatctgaagatggcagatttgaaaaaggtagcaacttcaaggtatctttcacaagaaccgtcgttat cagaacttatgtcaaatgtgaagatcaagcctattgaagaaaccccggtttcgccattggagttgattccagatatcgaaa tatcgactagaaagaaatacgatgcgtcgtgggatctgttgttccgtggtagaaaatataaatcgttcaacgattatgat SEQ ID NO: 41 DNA sequence of the K. lactis OCH1 gene: atggggttaccaaagatttcaagaagaacgaggtacattattgtcattgtgctgatactgtacttattgttttctgtgcaatg gaatactgcgaaagtgaatcaccatttctataacagcattggcacggtgcttcccagtacagctcgcgtggatcacttga acttgaaaaacttggacttagcaggtacgagcaataacggtgatcatttgatggatctacgagttcaattggctagtcaat tcccctacgattctcgagtacccatccccaaaaaggtatggcagacctggaagattgatcccagttcaaagtcacaggtt tcttccatttcaaaatgccagaatgattggaaacatttcagtgcatccgaggaaccgccatatcaataccaattaatcaca gatgatcaaatgataccacttctagagcagctatatggtggggtcccacaagtgataaaggcttttgaatccttgccactt ccaattcttaaagcagactttttcagatacttgatcctttatgcaagaggtggtatatattctgacatggatacgttcccatta aagccattgtcgtcatggccatcgacttctcagtcctacttttctagtttaaagaatccacaaaggtatagaaattccttgga caaccttgaaacgctagaagcttcagaacctggctttgtcattggtatcgaggctgatccggatagaagcgattgggca gagtggtacgccaggagaatacaattctgtcagtggacaatacaatcaaaatctggccatccattattgcgcgaattgat cacaaatatcaccgcaacaacattggagagcgtggcaaatgttaaaagcagcattcctttagatgatgctgaagtattaa aagacattgcagatgattataatgtcaacatgagggataaaaagaagttcaacaaaaattacaaacatcaacaaaagaa aaccgccaaaaatacagatggtaccgatattatgaactggactggtccaggtattttttcagatgttattttccagtatctta ataacgttatccagaagaatgatgacattttaattttcaatgataatcttaatgttatcaacaaacatggatccaaacatgata caactatgagattctataaagacattgttaaaaatttacaaaacgacaaaccctcattgttctggggattcttttcattgatga cagagcctattctagtggacgacatcatggtacttccgattacttctttctcaccaggtatcagaacaatgggcgctaaag aagacaacgacgagatggcatttgttaagcatatttttgaaggaagttggaaagactga SEQ ID NO: 42 Translation of putative K. lactis OCH1 gene: MGLPKISRRTRYIIVIVLILYLLFSVQWNTAKVNHHFYNSIGTVLPSTARVD HLNLKNLDLAGTSNNGDHLMDLRVQLASQFPYDSRVPIPKKVWQTWKID PSSKSQVSSISKCQNDWKHFSASEEPPYQYQLITDDQMIPLLEQLYGGVPQ VIKAFESLPLPILKADFFRYLILYARGGIYSDMDTFPLKPLSSWPSTSQSYFS SLKNPQRYRNSLDNLETLEASEPGFVIGIEADPDRSDWAEWYARRIQFCQW TIQSKSGHPLLRELITNITATTLESVANVKSSIPLDDAEVLKDIADDYNVNM RDKKKFNKNYKHQQKKTAKNTDGTDIMNWTGPGIFSDVIFQYLNNVIQK NDDILIFNDNLNVINKHGSKHDTTMRFYKDIVKNLQNDKPSLFWGFFSLMT EPILVDDIMVLPITSFSPGIRTMGAKEDNDEMAFVKHIFEGSWKDZ SEQ ID NO: 43 DNA sequence of the K. lactis MNN1 gene: atgatggttgtaaggcgttttttatcagcttcgcgatataatatgccatcttttaggtccaggcccgttctcttagctatctttg gtgtctgtgctaccgtgatatggtacctattctttttccagtctaatctgaagatggcagatttgaaaaaggtag caacttcaaggtatctttcacaagaaccgtcgttatcagaacttatgtcaaatgtgaagatcaagcctattgaagaaaccc cggtttcgccattggagttgattccagatatcgaaatatcgactagaaagaaatacgatgcgtcgtgggatctgttgttcc gtggtagaaaatataaatcgttcaacgattatgatcttcatacgaaatgtgagttttatttccagaatttatacaatttgaacg aggattggaccaataatattcggacgttcactttcgatattaacgatgtagacacgtctacgaaaattgacgctcttaaag attccgatggggttcaattggtggacgagaaggctatacgtttatacaagagaacgcataacgttgccttggctacgga aaggttacgtctttatgataaatgttttgtcaatagtccaggttcaaacccattgaaaatggatcaccttttcagatcgaaca agaagagtaagactacggctttggatgacgaagtcactgggaaccgtgatacttttaccaagacgaagaaaacttcgtt cttaagcgatatggacacgagtagtttccagaagtacgatcaatgggatttcgaacatagaatgttccccatgatcccata tttcgaggaacacaatttcaccaacgtgatgcctattttcaccggctcaaacggtggggaacctttacctcaagggaaatt cccggtattagatccaaaatccggtgaattgttacgtgtagagactttcagatatgataaatcgaaatcgctttggaagaa ctggaatgatatgtcctctgcttctggtaaacgtggtattatcttggctgctggcgacggccaagtggaccaatgcatcc gtcttattgctacgttgagagctcaaggaaacgctctacctattcaaattatccacaacaaccaattgaatgagaaatctgt gaaactgttatcggaggccgctaaatctaccgaattctcatccggtagagctcaatctctttggttagtgaatgtgggccc cacgttggaatcttcaatgaagagcaattttgggagatttaagaataagtggttgtcagttattttcaacacttttgaagaatt tatattcatagatacagatgccatctcctacattaatatggctgattatttcaacttcaaggagtacaaatctactggaacact cttctttaaggataggtctttggcaattggaactgaacagaaatgtggtcctttgttcgaaactcttgaaccaagaattcttg aaatgtactatttcaatactttacctatgatcaatggtgattacgtggaacagcaatgtatgggcatgctcaccccagagg aaaaagtttacaaacgtttctttgaagttggtcatcaacacaacttggaaagtggattattggccatcaacaaaaacgaac acatcatgggattggttactgcaacagtcttaaatatcgcaccaaaggtcggaggttgcggttggggtgacaaagagttt ttctggcttggtttgttggttgctggccaacgctactcgatctatgatatagatgcaagtgcaattggtgttcctcaacagaa gcaatctatcgctaacggagacgaatttgatgaatataggatttgttctttacaagtggcacatacttcatacgacggacat ttactatggataaatggtggctctcagtactgtaagaaaccagagacttttgaaggtgattggaccaacattaaggagctt cgtgaatcgtattctgatgataaagaaaaggctctgaaggcttatagtgatacagttaaggtggaagcagcaatcgtgc cagattccagaagtaatggttggggtagagacgatcaaagatgtaaaggctacttctggtgcggcaaatttacttcaaa gctgaaaccgtatacttataacacggtggtaactaaaggtgatttgatccgtttcggagacgaggaaatcgaaagtatct ccaagattaataagatctggaatgatgctattattccagacggagcttaa SEQ ID NO: 44 Translation of putative K. lactis MNN1 gene: MMVVRRFLSASRYNMPSFRSRPVLLAIFGVCATVIWYLFFFQSNLKMADL KKVATSRYLSQEPSLSELMSNVKIKPIEETPVSPLELIPDIEISTRKKYDASW DLLFRGRKYKSFNDYDLHTKCEFYFQNLYNLNEDWTNNIRTFTFDINDVD TSTKIDALKDSDGVQLVDEKAIRLYKRTHNVALATERLRLYDKCFVNSPG SNPLKMDHLFRSNKKSKTTALDDEVTGNRDTFTKTKKTSFLSDMDTSSFQ KYDQWDFEHRMFPMIPYFEEHNFTNVMPIFTGSNGGEPLPQGKFPVLDPKS GELLRVETFRYDKSKSLWKNWNDMSSASGKRGIILAAGDGQVDQCIRLIA TLRAQGNALPIQIIHNNQLNEKSVKLLSEAAKSTEFSSGRAQSLWLVNVGP TLESSMKSNFGRFKNKWLSVIFNTFEEFIFIDTDAISYINMADYFNFKEYKST GTLFFKDRSLAIGTEQKCGPLFETLEPRILEMYYFNTLPMINGDYVEQQCM GMLTPEEKVYKRFFEVGHQHNLESGLLAINKNEHIMGLVTATVLNIAPKV GGCGWGDKEFFWLGLLVAGQRYSIYDIDASAIGVPQQKQSIANGDEFDEY RICSLQVAHTSYDGHLLWINGGSQYCKKPETFEGDWTNIKELRESYSDDKE KALKAYSDTVKVEAAIVPDSRSNGWGRDDQRCKGYFWCGKFTSKLKPYT YNTVVTKGDLIRFGDEEIESISKINKIWNDAIIPDGA SEQ ID NO: 45-56: See Table 12. Primer name Primer sequence NeuA sense 5′-ATGAGAACAAAAATTATTGCGATAATTCCAGCCCG-3′ (SEQ ID NO: 45) NeuA antisense 5′-TCATTTAACAATCTCCGCTATTTCGTTTTC-3′ (SEQ ID NO: 46) NeuB sense 5′-ATGAGTAATATATATATCGTTGCTGAAATTGGTTG-3′ (SEQ ID NO: 47) NeuB antisense 5′-TTATTCCCCCTGATTTTTGAATTCGCTATG-3′ (SEQ ID NO: 48) NeuC sense 5′-ATGAAAAAAATATTATACGTAACTGGATCTAGAG-3′ (SEQ ID NO: 49) NeuC antisense 5′-CTAGTCATAACTGGTGGTACATTCCGGGATGTC-3′ (SEQ ID NO: 50) mouse CMP-Sia 5′-ATGGACGCGCTGGAGAAGGGGGCCGTCACGTC-3′ (SEQ ID NO: 51) synthase sense mouse CMP-Sia 5′-CTATTTTTGGCATGAGTTATTAACTTTTTCTATCAG-3′ (SEQ ID NO: 52) synthase antisense porcine GlcNAc 5′-ATGGAGAAGGAGCGCGAAACTCTGCAGG-3′ (SEQ ID NO: 53) epimerase sense porcine GlcNAc 5′-CTAGGCGAGGCGGCTCAGCAGGGCGCTC-3′ (SEQ ID NO: 54) epimerase antisense E. coli Sialate 5′-ATGGCAACGAATTTACGTGGCGTAATGGCTG-3′ (SEQ ID NO: 55) aldolase sense E. coli Sialate 5′-TCACCCGCGCTCTTGCATCAACTGCTGGGC-3′ (SEQ ID NO: 56) aldolase antisense SEQ ID NO: 57-68: Are disclosed throughout the specification. SEQ ID NO: 69-76: See Table 12. mouse bifunctional 5′-ATGGAGAAGAACGGGAACAACCGAAAGCTCCG-3′ (SEQ ID NO: 69) UDP-N- acetylglucosamine- 2-epimerase/N- acetylmannosamine kinase sense mouse bifunctional 5′-CTAGTGGATCCTGCGCGTTGTGTAGTCCAG-3′ (SEQ ID NO: 70) UDP-N- acetylglucosamine- 2-epimerase/N- acetylmannosamine kinase antisense mouse Sia9P syn 5′-ATGCCGCTGGAACTGGAGCTGTGTCCCGGGC-3′ (SEQ ID NO: 71) sense mouse Sia9P syn 5′-TTAAGCCTTGATTTTCTTGCTGTGACTTTCCAC-3′ (SEQ ID NO: 72) antisense human N- 5′-GGGAGAATGCGGCCGCCACCATGGGGCTGAGCCGC GTGCGGGCG GTTTTC-3′ acetylneuraminic (SEQ ID NO: 73) acid phosphatase   (NANP) sense human N- 5′-GTATAGACTGCAAAGTCAGTATGTCCACTTGATTAATTAACC-3′ (SEQ ID NO: 74) acetylneuraminic acid phosphatase (NANP) antisense human CMP-Sia 5′-ATGGACTCGGTGGAGAAGGGGGCCGCCACC-3′ (SEQ ID NO: 75) synthase sense human CMP-Sia 5′-CTATTTTTGGCATGAATTATTAACTTTTTCC-3′ (SEQ ID NO: 76) synthase antisense SEQ ID NO: 77 Primer for hGNE: GGGAGAATGCGGCCGCCACCATGGAGAAGAATGGAAATAACCGAAAGCTGCG SEQ ID NO: 78 Primer for hGNE: CCTTAATTAACTAGTAGATCCTGCGTGTTGTGTAGTCCAGAAC SEQ ID NO: 79 Primer for hSiaPsyn GGGAGAATGCGGCCGCCACCATGCCGCTGGAGCTGGAGCTGTGTCCCG SEQ ID NO: 80 Primer for hSiaPsyn CCTTAATTAATTAAGACTTGATTTTTTTGCCATGATTATCTACC SEQ ID NO: 81 Primer GGCTCGAGATTTAAATGCGTACCTCTTCTACGAGATTC SEQ ID NO: 82 Primer CCCTCGAGATTTAAATCCAACCGATAAGGTGTACAGGAG SEQ ID NO: 83 Primer GGCTCGAGCGGCCGCCACCATGAATAGCATACACATGAACGCCAATACG SEQ ID NO: 84 Primer CCCTCGAGTTAATTAACTAGACGCGCGGCAGCAGCTTCTCCTCATCG-3′) SEQ ID NO: 85 Primer ATG ACT GGT GTT CAT GAA GGG SEQ ID NO: 86 Primer TTA CTT ATA TGT CTT GGT ATG SEQ ID NO: 87 Primer GCG GCC GCA TGA CTG GTG TTC ATG AAG GGA CTG TGT TGG TTA CTG GCG GCG CTG GTT ATA TAG GTT CTC ATA CGT GCG TTG TTT TGT TAG AAA A SEQ ID NO: 88 Primer TTA ATT AAT TAC TTA TAT GTC TTG GTA TG 3′) SEQ ID NO: 89 Primer GCCGCGACCTGAGCCGCCTGCCCCAAC SEQ ID NO: 90 Primer CTAGCTCGGTGTCCCGATGTCCACTGT SEQ ID NO: 91 Primer CTTAGGCGCGCCGGCCGCGACCTGAGCCGCCTGCCC SEQ ID NO: 92 Primer GGGGCATATCTGCCGCCCATC SEQ ID NO: 93 Primer GATGGGCGGCAGATATGCCCC SEQ ID NO: 94 Primer CTTCTTAATTAACTAGCTCGGTGTCCCGATGTCCAC SEQ ID NO: 95 Primer GGGAGAATGCGGCCGCCACCATGGACTCGGTGGAGAAGGGGGCCGCCACCTC SEQ ID NO: 96 Primer CCTTAATTAACTATTTTTGGCATGAATTATTAACTTTTTCCATTA SEQ ID NO: 97 Primer CGGAATTCCACCATGGCTCCGGCGAGAGAAAATGTCAG SEQ ID NO: 98 Primer CGGAATTCTCACACACCAATGATTCTCTCTTTTGAAG SEQ ID NO: 99 Primer GGCTCGAGATTTAAATGCGTACCTCTTCTACGAGATTC SEQ ID NO: 100 Primer CCCTCGAGATTTAAATCCAACCGATAAGGTGTACAGGAG SEQ ID NO: 101 Primer GGCGCGCCAGCAAGCAAGACCCTAAGGAAGACATTCC SEQ ID NO: 102 Primer CCTTAATTAATCAACAACGAATGTTCCGGAAGCCAGAAAGG SEQ ID NO: 103 and 104 Nucleic acid (SEQ ID NO: 103) and amino acid sequence (SEQ ID NO: 104) of the codon-optimized hST6Gal leader 53 fusion (FIG. 33)

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1-56. (canceled)
 57. A method for producing CMP-sialic acid in a yeast host cell comprising: (a) transforming into the yeast host cell one or more nucleic acid molecules encoding the enzymes of a sialylation pathway consisting of E. coli NeuC, E. coli NeuB, and a CMP-Sialic acid synthase; and (b) growing the yeast host cell under conditions effective for producing the CMP-sialic acid.
 58. The method of claim 57, wherein the yeast host cell is a Pichia sp., Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica; Saccharomyces sp., Saccharomyces cerevisiae, Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, or Candida albicans.
 59. The method of claim 57, wherein the yeast host cell is Pichia pastoris.
 60. A yeast host cell capable of producing CMP-sialic acid comprising: a sialylation pathway consisting of E. coli NeuC, E. coli NeuB, and a CMP-Sialic acid synthase, wherein the yeast host cell is capable of producing the CMP-sialic acid.
 61. The yeast host cell of claim 60, wherein the yeast host cell is a Pichia sp., Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica; Saccharomyces sp., Saccharomyces cerevisiae, Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, or Candida albicans.
 62. The yeast host cell of claim 60, wherein the yeast host cell is Pichia pastoris.
 63. A method for producing CMP-sialic acid comprising: (a) providing a yeast host cell comprising a CMP-sialic acid biosynthetic pathway consisting of E. coli NeuC, E. coli NeuB, and a CMP-Sialic acid synthase; and (b) growing the yeast host cell under conditions effective for producing the CMP-sialic acid.
 64. The method of claim 63, wherein the yeast host cell is a Pichia sp., Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica; Saccharomyces sp., Saccharomyces cerevisiae, Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, or Candida albicans.
 65. The method of claim 63, wherein the yeast host cell is Pichia pastoris. 